bluuebunny/crossref_metadata_2025 huggingface dataset:
Supplementary Tables S3 from CircMMP2(6,7) Cooperates with β-Catenin and PRMT5 to Disrupt Bone Homeostasis and Promote Breast Cancer Bone Metastasis
Authors: Xu Yingru , Li Xincheng , Zhang Shuxia , Tang Miaoling , Yu Ruyuan , Liao Xinyi , Li Ziwen , Li Man , Chen Suwen , Qian Wanying , Song Libing , Ke Zunfu , Li Jun
Published: January 2024

Primers, Probes, siRNA, shRNA and ASOs oligonucleotides
Supplementary Table 3 from Small Cell Lung Cancer Plasticity Enables NFIB-Independent Metastasis
Authors: Ko Julie H. , Lambert Kyle E. , Bhattacharya Debadrita , Lee Myung Chang , Colón Caterina I. , Hauser Haley , Sage Julien
Published: January 2024

Enrichr analysis RNA-seq
Supplementary Tables S2 from CircMMP2(6,7) Cooperates with β-Catenin and PRMT5 to Disrupt Bone Homeostasis and Promote Breast Cancer Bone Metastasis
Authors: Xu Yingru , Li Xincheng , Zhang Shuxia , Tang Miaoling , Yu Ruyuan , Liao Xinyi , Li Ziwen , Li Man , Chen Suwen , Qian Wanying , Song Libing , Ke Zunfu , Li Jun
Published: January 2024

Univariate and Multivariate Analyses of Various Prognostic Parameters in Breast Cancer Patients by Cox-regression Analysis
Supplementary Figure 1 from Small Cell Lung Cancer Plasticity Enables NFIB-Independent Metastasis
Authors: Ko Julie H. , Lambert Kyle E. , Bhattacharya Debadrita , Lee Myung Chang , Colón Caterina I. , Hauser Haley , Sage Julien
Published: January 2024

NFIB upregulation correlates with SCLC tumor progression in RPR2 mutant mice
Data from Glucose Deprivation Promotes Pseudohypoxia and Dedifferentiation in Lung Adenocarcinoma
Authors: Saggese Pasquale , Pandey Aparamita , Alcaraz Martín , Fung Eileen , Hall Abbie , Yanagawa Jane , Rodriguez Erika F. , Grogan Tristan R. , Giurato Giorgio , Nassa Giovanni , Salvati Annamaria , Shirihai Orian S. , Weisz Alessandro , Dubinett Steven M. , Scafoglio Claudio
Published: January 2024

Abstract
Increased utilization of glucose is a hallmark of cancer. Sodium-glucose transporter 2 (SGLT2) is a critical player in glucose uptake in early-stage and well-differentiated lung adenocarcinoma (LUAD). SGLT2 inhibitors, which are FDA approved for diabetes, heart failure, and kidney disease, have been shown to significantly delay LUAD development and prolong survival in murine models and in retrospective studies in diabetic patients, suggesting that they may be repurposed for lung cancer. Despite the antitumor effects of SGLT2 inhibition, tumors eventually escape treatment. Here, we studied the mechanisms of resistance to glucose metabolism-targeting treatments. Glucose restriction in LUAD and other tumors induced cancer cell dedifferentiation, leading to a more aggressive phenotype. Glucose deprivation caused a reduction in alpha-ketoglutarate (αKG), leading to attenuated activity of αKG-dependent histone demethylases and histone hypermethylation. The dedifferentiated phenotype depended on unbalanced EZH2 activity that suppressed prolyl-hydroxylase PHD3 and increased expression of hypoxia-inducible factor 1α (HIF1α), triggering epithelial-to-mesenchymal transition. Finally, a HIF1α-dependent transcriptional signature of genes upregulated by low glucose correlated with prognosis in human LUAD. Overall, this study furthers current knowledge of the relationship between glucose metabolism and cell differentiation in cancer, characterizing the epigenetic adaptation of cancer cells to glucose deprivation and identifying targets to prevent the development of resistance to therapies targeting glucose metabolism.
Significance:
Epigenetic adaptation allows cancer cells to overcome the tumor-suppressive effects of glucose restriction by inducing dedifferentiation and an aggressive phenotype, which could help design better metabolic treatments.
Data from Spatial and Single-Cell Transcriptomics Reveal a Cancer-Associated Fibroblast Subset in HNSCC That Restricts Infiltration and Antitumor Activity of CD8+ T Cells
Authors: Li Chuwen , Guo Haiyan , Zhai Peisong , Yan Ming , Liu Chun , Wang Xiaoning , Shi Chaoji , Li Jiang , Tong Tong , Zhang Zhiyuan , Ma Hailong , Zhang Jianjun
Published: January 2024

Abstract
Although immunotherapy can prolong survival in some patients with head and neck squamous cell carcinoma (HNSCC), the response rate remains low. Clarification of the critical mechanisms regulating CD8+ T-cell infiltration and dysfunction in the tumor microenvironment could help maximize the benefit of immunotherapy for treating HNSCC. Here, we performed spatial transcriptomic analysis of HNSCC specimens with differing immune infiltration and single-cell RNA sequencing of five pairs of tumor and adjacent tissues, revealing specific cancer-associated fibroblast (CAF) subsets related to CD8+ T-cell infiltration restriction and dysfunction. These CAFs exhibited high expression of CXCLs (CXCL9, CXCL10, and CXCL12) and MHC-I and enrichment of galectin-9 (Gal9). The proportion of MHC-IhiGal9+ CAFs was inversely correlated with abundance of a TCF1+GZMK+ subset of CD8+ T cells. Gal9 on CAFs induced CD8+ T-cell dysfunction and decreased the proportion of tumor-infiltrating TCF1+CD8+ T cells. Collectively, the identification of MHC-IhiGal9+ CAFs advances the understanding of the precise role of CAFs in cancer immune evasion and paves the way for more effective immunotherapy for HNSCC.
Significance:
Spatial analysis identifies IFN-induced MHC-IhiGal9+ CAFs that form a trap for CD8+ T cells, providing insights into the complex networks in the tumor microenvironment that regulate T-cell infiltration and function.
Abstract A027: Aberrant glycosylation in mPDAC cells is associated with increased recruitment of immunosuppressive cells
Authors: Brookens Shawna , Degaramo David , Wilson Kiesha , Obregon Wilder Condori , Nagarkatti Mitzi , Posey Avery
Published: January 2024

Tumor associated-glycan Tn antigen (aGalNAc-a-O-Ser/Thr) is associated with poor prognosis in various solid tumors. However, the mechanisms that Tn antigen promotes tumorigenesis is incompletely understood. Here, we generated Tn antigen-expressing murine pancreatic ductal adenocarcinoma (mPDAC) tumor cell lines through CRISPR/Cas9-mediated knockout of the C1galt1c1 gene. Tn antigen expressing mPDAC cell lines exhibited increased in vitro proliferation and migration compared to parental controls. RNA sequencing revealed that loss of C1galt1c1 in mPDAC cells correlated with increased expression of myeloid recruitment and differentiation genes, IL34 and Lif. Additionally, C1galt1c1 knockout mPDAC cells implanted into mice led to accelerated tumor growth and increased tumor infiltration of myeloid-derived suppressor cells and neutrophils as determined by flow cytometry and scRNAseq. Interestingly, surface expression of a binding partner of Tn antigen, macrophage galactose N-acetyl galactosamine specific lectin 2 (Mgl2), was decreased in the infiltrate of C1galt1c1 knockout tumors suggesting a role for the Mgl2-Tn interaction in tumor progression. Together, our data indicate that expression of Tn antigen on tumor cells promotes an immunosuppressive microenvironment in mPDAC.

Citation Format: Shawna Brookens, David Degaramo, Kiesha Wilson, Wilder Condori Obregon, Mitzi Nagarkatti, Avery Posey Jr.. Aberrant glycosylation in mPDAC cells is associated with increased recruitment of immunosuppressive cells [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 A027.
Abstract A106: Single cell RNA sequencing of EPCAM+ cells reveals unique malignant biology
Authors: Arbe-Barnes Edward H. , Jainarayanan Ashwin , Abu-Shah Enas , Bashford-Rogers Rachael J.M. , Sivakumar Shivan
Published: January 2024

Pancreatic ductal adenocarcinoma (PDAC) is notable due to its complex and dense stroma which comprises a high proportion of non-malignant cells. Studying PDAC epithelial cells at the single cell level may yield insights which have not been appreciated through bulk tissue analysis. We analysed a novel single cell RNA sequencing (scRNA seq) dataset of 11000 epithelial (EPCAM+) cells from 7 resected PDAC samples and used a validation cohort of 43 samples from two published scRNA seq datasets. Malignant epithelial cells were identified by: gene expression, changes in copy number, and by the presence of known PDAC single nucleotide variants. This confirmed that sorting for EPCAM+ cells prior to scRNA seq resulted in an enriched proportion of malignant vs non-malignant tumor epithelial cells. This dataset of enriched single cell dataset was therefore useful for studying the influence of intrinsic malignant cell factors on PDAC biology. We observed that malignant cells do not segregate into classical or basal-like signatures, despite these being clinically validated bulk transcriptomic tumor subtypes. Indeed, classical and basal-like signatures module scores in PDAC in individual malignant cells were positively correlated, suggesting they are not divergent programs in malignant cells. Bulk transcriptomic signature subtypes are therefore most likely to reflect differences in PDAC tissue stroma, rather than in intrinsic malignant cell factors. Moreover, we sort to investigate intra- and inter- sample heterogeneity to understand the intrinsic malignant cell factors which may influence the biology of PDAC. We analysed single cell velocity to understand the dynamics of malignant cell states. This showed heterogeneity in expression of known pan-cancer malignant cell meta-programs which identified a population of cells with high expression of cell cycle genes, suggesting greater proliferation. We then explored expression of immune modulating genes to determine how malignant cells influence the immunosuppressive microenvironment. For example, we identified heterogeneity in HLA class 1 and antigen presentation gene expression which may be a mechanism of immune suppression in PDAC. Together these data demonstrate how studying scRNA seq of EPCAM+ cells reveals intrinsic malignant cell factors which cannot be appreciated through bulk RNA seq.

Citation Format: Edward H. Arbe-Barnes, Ashwin Jainarayanan, Enas Abu-Shah, Rachael J.M. Bashford-Rogers, Shivan Sivakumar. Single cell RNA sequencing of EPCAM+ cells reveals unique malignant biology [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 A106.
Abstract B006: Exosomal protein Eps8 as a new biomarker for pancreatic cancer
Authors: Hayashi Kazunao , Tsuchiya Atsunori , Terai Shuji
Published: January 2024

Exosomes are extracellular vesicles containing nucleic acids, proteins, and lipids of approximately 100 nm, which are released from cancer cells to create a microenvironment favorable for their survival. Epidermal growth factor receptor pathway substrate 8 (Eps8), an exosomal protein, is reportedly associated with metastasis. We investigated the relationship between Eps8 in the serum exosomes of patients with pancreatic cancer and clinicopathological factors and examined the usefulness of exosomal Eps8 as a biomarker for pancreatic cancer. Methods: Serum exosomes of 93 patients with pancreatic disease (64 cases of pancreatic cancer, 12 cases of chronic pancreatitis, and 17 cases of IPMN) were extracted via ultracentrifugation, and Eps8 in exosomes were evaluated by western blotting. We analyzed 1) the clinicopathological factors of patients with pancreatic cancer, 2) the Eps8 positive rate in each disease, 3) the sensitivity, specificity, and positive predictive value of Eps8 for the diagnosis of pancreatic cancer, 4) the relationship between Eps8 and clinicopathological factors, and 5) compared and discussed between exosomal Eps8 and serum CA19-9. Results: The median patient age was 72 years (47–85), with 35 male and 29 female patients included in the study. The median body mass index was 20.8 kg/m2 (12–26.7 kg/m2). 1) Site Ph33/Pbt31, cStageIV/III/II/I: 28/11/14/11; 2) pancreatic cancer 59.4%, chronic pancreatitis 33%, IPMN 12%; 3) sensitivity 59.4. 4) In patients with pancreatic cancer, Eps8-positive patients had significantly higher ALT (p = 0.03), significantly less cancer (p = 0.04), lower IPMN complication rates, and more chronic pancreatitis and autoimmune diseases than the exosomal Eps8-negative patients. Stage I 64% (7/11), Stage II 36% (5/14), Stage III 64% (7/11) Stage IV 68% (19/28) 5) Exosomal Eps8 positivity (64%) was higher than serum CA19-9 positivity (45%) in Stage I. We also found some patients were negative for serum CA19-9 but positive for exosomal Eps8. Conclusion: Exosomal Eps8 has a higher positive rate in Stage I than serum CA19-9. Diagnosis with exosomal Eps8 may improve diagnostic performance for pancreatic cancer.

Citation Format: Kazunao Hayashi, Atsunori Tsuchiya, Shuji Terai. Exosomal protein Eps8 as a new biomarker for pancreatic cancer [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 B006.
Abstract B016: Eosinophils alter metastatic spread in pancreatic cancer
Authors: Hoffman Megan T. , Lenehan Patrick J. , Wang Jen , Ali Lestat , Uchida Amiko , Vayrynen Sara , Costa Andressa Dias , Dougan Michael , Heckler Max , Nowak Jonathan , Wolpin Brian W. , Dougan Stephanie K.
Published: January 2024

Pancreatic ductal adenocarcinoma (PDA) is the third most common cause of cancer death in the US with a 11% 5-year survival rate. A key contributor of this dismal prognosis is early and frequent metastatic spread with limited treatment options for patients. Metastasis in PDA is facilitated by a robust fibroinflammatory response in the primary tumor site, a large portion of which is composed of infiltrating immune cells, and survival during transit to establish in the secondary site. In the primary tumor reactive stroma, we found increased eosinophils in both human and mouse PDA compared to the normal pancreas. Eosinophils are an innate immune cell best known for their ability to control parasite responses and have been implicated in either pro- or anti-cancer roles in various solid tumors. We used the ΔdblGata mice, which lack eosinophils, to study the role of eosinophils during metastatic spread using a resectable model of PDA metastasis. This model of metastasis was developed to facilitate consistent seeding of lung metastasis in approximately 50 percent of mice from a transient subcutaneous tumor which undergoes subsequent surgical removal and harvest to evaluate metastasis at 4 weeks. We found that mice lacking eosinophils have increased metastasis to the lung in our resectable model of PDA metastasis but no difference in establishment of IV injected PDA tumor cells, suggesting eosinophil control at the primary tumor site. Single cell sequencing of the subcutaneous tumors finds differences in the angiogenic subtypes, tip and stalk cells, in mice lacking eosinophils. Sequencing of eosinophil populations in humans and mice find an increase in angiogenic signaling suggesting a regulatory role in tumor cell access to the circulation. We conclude that eosinophils in PDA inhibit the metastatic process through control of angiogenic signaling.

Citation Format: Megan T. Hoffman, Patrick J. Lenehan, Jen Wang, Lestat Ali, Amiko Uchida, Sara Vayrynen, Andressa Dias Costa, Michael Dougan, Max Heckler, Jonathan Nowak, Brian W. Wolpin, Stephanie K. Dougan. Eosinophils alter metastatic spread in pancreatic cancer [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 B016.
Abstract B020: Charting the cellular heterogeneity of primary and metastatic PDAC microenvironment
Authors: Blanco-Gomez Adrian , Felton Catherine , Banyard Antonia , Jorgensen Claus
Published: January 2024

Cancer-associated fibroblasts (CAFs) are important regulators of tumor progression and therapeutic response in PDAC, with several functionally diverse populations now identified. However, while CAF populations with similar transcriptional characteristics have been identified across different tissues, emerging data suggests that these populations may diverge functionally. For example, whereas antigen presenting CAFs (apCAFs) have been suggested to promote immune evasion in PDAC, apCAFs have been associated with anti-tumor immunity in lung cancer. Thus, high resolution mapping of the microenvironment in both primary and metastatic tumors is required to define tissue specific and conserved subsets for functional interrogation. We deployed cytometry by time of flight (CyTOF) to annotate stromal and immune subsets in healthy and tumor bearing murine pancreas, liver and lung. Spatial proximity of tumor and host cells was further annotated using sLP-mCherry, a secreted lipo-permeable form of mCherry. In total, we annotated 50.3 million cells across normal and tumor bearing tissues. Stromal fibroblast populations were highly divergent across both healthy and tumor bearing tissues. Commonly used markers were expressed in a manner dominated by tissue of origin, with less than 10% of fibroblasts and CAFs displaying common phenotypes across tissues. For example, aSMApos/PDGFRalow myCAFs were abundant in lung and liver, Ly6cpos/PDGFRapos/aSMAlow iCAFs were more abundant in the pancreas and MHCIIpos/CD74pos apCAFs were more dominant in lung. In contrast, most immune subsets, with exception of alveolar macrophages and Kupfer cells, were consistently observed across all tissues analysed albeit at different abundances. Tumors arising from epithelial and mesenchymal PDAC cells induce unique changes in CAF subset abundances where epithelial tumors are enriched for myCAFs and mesenchymal tumors induce more apCAFs. These observations were consistent across tumor bearing liver and lung. MyCAFs and PD-L1 or PDL-2 expressing macrophages were generally more proximal to epithelial tumor cells, whereas tumors originating from mesenchymal PDAC cells were enriched for antigen-experienced T cells. Taken together these analyses reveal tissue specific effects on the emerging microenvironment, with shared properties emerging from immune and mesenchymal niche composition.

Citation Format: Adrian Blanco-Gomez, Catherine Felton, Antonia Banyard, Claus Jorgensen. Charting the cellular heterogeneity of primary and metastatic PDAC microenvironment [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 B020.
Abstract B031: Targeting epigenetic machinery to revoke stromal pro-tumorigenicity and enhance efficacy in pancreatic cancer therapy
Authors: Liang Gaoyang , Johnson Jeremiah A. , Evans Ronald M.
Published: January 2024

Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) induce prominent desmoplasia in part by activating fibroblasts or their progenitors in the stroma. The resultant cancer-associated fibroblasts (CAFs) often switch on the pro-desmoplastic, pro-inflammatory and/or pro-tumorigenic transcriptional programs, contributing to tumor progression and therapeutic resistance. Identifying and targeting epigenetic machinery essential for stromal fibroblast activation provides potential strategies to revoke stromal pro-tumorigenicity and overcome therapeutic resistance in PDAC. Here we identify Class I histone deacetylases (HDACs) as key epigenetic regulators facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in stromal fibroblasts. Mechanistically, HDACs coordinate the establishment of chromatin architecture that enables the activation of pro-desmoplastic programs directed by the transcriptional factor axis of serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also facilitate the induction of pro-inflammatory program in stromal fibroblasts, supporting leukemia inhibitory factor (LIF)-mediated pro-tumorigenic crosstalk. Both HDAC depletion in CAFs and treatment of the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a novel lipogenic fibroblast subpopulation in CAFs, a potential precursor population for activated myofibroblasts in vivo. Our work pinpoints a critical role of HDACs in driving stromal pro-tumorigenicity and highlights the stromal targeting capacity of HDACi therapy. Furthermore, the clinical utility of HDACi therapy is often hindered by limited potency and nonnegligible systemic toxicity of the HDAC inhibitors. To facilitate the translation of HDACi-based therapy, we develop a polymer-based bottlebrush pro-drug (BPD) nanoparticle incorporating Ent. The Ent-BPD nanoparticle overcomes the limitations of conventional HDACi therapy by sustaining long-lasting release of HDACi activity specifically at the tumor sites. Administration of Ent-BPD in PDAC mouse models achieves superior intratumoral HDACi potency and enhanced therapeutic efficacy compared to the free drug Ent treatment; meanwhile, it avoids the systemic toxicity associated with the free drug treatment. Our work further demonstrates a promising strategy for the clinical adaptation of HDACi-based therapy.

Citation Format: Gaoyang Liang, Jeremiah A. Johnson, Ronald M. Evans. Targeting epigenetic machinery to revoke stromal pro-tumorigenicity and enhance efficacy in pancreatic cancer therapy [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 B031.
Abstract B034: Antigen-presenting cancer-associated fibroblasts are found in immunotherapy-sensitive murine models of pancreatic ductal adenocarcinoma
Authors: Maru Saumya , Howe Kathryn , Andaloori Lalitya , Leatherman James , Tandurella Joseph , Pearce Edward J. , Jaffee Elizabeth M.
Published: January 2024

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths. Despite the advent of immune checkpoint inhibition (ICI) that has markedly changed the treatment landscape for select tumors with respect to both outcomes and tolerability, PDAC remains unresponsive to such an approach. One feature of the PDAC tumor microenvironment (TME) that remains poorly understood is the interaction between cancer-associated fibroblasts (CAFs) and immune and tumor cells. Unique subsets of CAFs have been identified, including antigen-presenting CAFs (apCAFs) which are defined by expression of MHC-II and can activate CD4 T cells in genetically engineered mouse models of PDAC. apCAFs are rarely found in human PDAC; in a human PDAC atlas consisting of six published human single cell RNA-sequencing (scRNA-seq) datasets, we identified 370 apCAFs out of 8736 total CAFs, the majority of which were found in one dataset. Here, we have orthotopically implanted cell lines derived from the autochthonous KPC murine model of PDAC which mimics the stromal components of human disease to further study the influence of apCAFs on the anti-tumor response. We have found that although the overall CAF population is equivalent between tumors, apCAFs are present primarily in tumors derived from KPC cell lines that display ICI sensitivity. apCAFs sorted from these tumors and pulsed with OVA peptides upregulated CD25 and CD69 expression on naïve OT-II CD4 T cells, confirming that apCAFs in our system can present via MHC-II. Furthermore, these apCAFs increased CD4 activation markers upon ex-vivo coculture with dissociated KPC tumors. We therefore hypothesize that apCAFs are required for the ICI-mediated anti-tumor effect and do so via modulation of CD4 T cell responses. Although a small proportion of the total compartment, the apCAF subset may be enhanced in number and function by CAF-targeted modulators to synergize with ICI to affect the antitumor response, which could provide a solution to the currently unmet need for new and more effective treatments for PDAC patients.

Citation Format: Saumya Maru, Kathryn Howe, Lalitya Andaloori, James Leatherman, Joseph Tandurella, Edward J. Pearce, Elizabeth M. Jaffee. Antigen-presenting cancer-associated fibroblasts are found in immunotherapy-sensitive murine models of pancreatic ductal adenocarcinoma [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 B034.
Abstract B039: Hypoxia promotes an inflammatory phenotype of fibroblasts in pancreatic cancer
Authors: Ngodup Tenzin , Mello Ashley
Published: January 2024

Pancreatic ductal adenocarcinoma (PDAC) is notable for its dense desmoplastic stroma, consisting of extracellular matrix, cancer-associated fibroblasts (CAFs), and immune cells. Another notable feature of the PDAC microenvironment is hypoxia, a condition of insufficient oxygen availability. Although hypoxia leads to adaptive responses in both cancer cells and stromal cells, the effects of hypoxia on the PDAC stroma and tumor-stroma interactions are not fully understood. Cancer-associated fibroblasts (CAFs) are a predominant and heterogeneous stromal cell type in PDAC, and single-cell transcriptomics of human and mouse PDAC has recently revealed a distinct CAF subpopulation, inflammatory CAFs (iCAFs). These inflammatory fibroblasts produce high levels of cytokines and chemokines in PDAC and have the potential to contribute to its immunosuppressive microenvironment and tumorigenesis. By injecting a hypoxia probe into PDAC mouse models, we recently found that iCAFs predominantly reside in hypoxic tumor regions. We also observed that the hypoxia-related gene signature is positively enriched in iCAFs in human PDAC samples. By exposing 3D cocultures of pancreatic tumor cells and fibroblasts to either hypoxia or normoxia, we demonstrated that hypoxia promotes iCAF formation via IL1ɑ secreted from tumor cells. Importantly, the presence of hypoxic fibroblasts further elevated IL1ɑ levels in tumor cells, implicating hypoxia as a modulator of bidirectional interactions between tumor cells and fibroblasts. Efforts are ongoing to understand the mechanism by which hypoxia promotes iCAF formation by modulating bidirectional interactions between tumor cells and fibroblasts in PDAC. Together, our study identifies hypoxia as a key environmental cue for inducing an iCAF phenotype, thus highlighting an instructive role of hypoxia in shaping the stromal microenvironment.

Citation Format: Tenzin Ngodup, Ashley Mello. Hypoxia promotes an inflammatory phenotype of fibroblasts in pancreatic cancer [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 B039.
Abstract B040: Neural control of cancer-associated fibroblasts in PDAC
Authors: Nigri Jeremy , Lan Wenjun , Chavan Sangeeta S , Park Youngkyu , Borniger Jeremy , Tracey Kevin J , Tuveson David A
Published: January 2024

Pancreatic ductal adenocarcinoma (PDAC) poses a formidable challenge to public health, with a dismal 5-year survival rate and a projected rise as the second leading cause of cancer-related deaths by 2030. The pathogenesis of PDAC involves a robust desmoplastic reaction and a complex tumor microenvironment (TME), constituting up to 80% of the tumor volume. The TME is a heterogeneous mixture of cells, including cancer-associated fibroblasts (CAFs), immune cells, endothelial cells, and neurons, which all interact and contribute to disease progression. However, the precise interplay between the tumor-associated nerves and PDAC development remains unclear. It is known that neural remodeling within the tumor microenvironment are closely associated with intense neuropathic pain, metastasis, and shortened patient survival. The prevalence of nerve fibers present in the dense desmoplastic stroma of PDAC tumors suggest a potential interaction between neurons and stromal cells, particularly CAFs, through various paracrine signaling mechanisms, influencing CAF behavior and facilitating neural remodeling, ultimately driving PDAC progression. Our investigation revealed that the neurotransmitter noradrenaline, induces calcium signaling and prompt contraction in pancreatic stellate cells (PSCs). Preliminary in vitro findings indicate that this calcium signaling is mediated by alpha 1 adrenergic receptors. Whole-mount 3D imaging mouse PDAC tissues, indicates a spatial organization of adrenergic neurons in immediate proximity to neoplastic cells and cancer-associated fibroblasts. Moreover, our study demonstrated that noradrenaline and alpha 1 agonists modulate extracellular matrix (ECM) production and cytokine secretion in PSCs and CAFs. Finally, we discovered that chemical ablation of the sympathetic nervous system significantly suppresses tumor growth while promoting metastasis, underscoring the critical role of the noradrenergic system in tumor development. Collectively, our findings propose that noradrenergic neurons possess the capacity to influence CAF behavior, opening new avenues of investigation to unravel the intricate crosstalk between neural regulation and tumor progression in PDAC.

Citation Format: Jeremy Nigri, Wenjun Lan, Sangeeta S Chavan, Youngkyu Park, Jeremy Borniger, Kevin J Tracey, David A Tuveson. Neural control of cancer-associated fibroblasts in PDAC [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 B040.
Abstract B049: Pancreatic beta cell stress pathways drive pancreatic ductal adenocarcinoma development in obesity
Authors: Garcia Cathy C. , Venkat Aarthi , Tong Alex , Agabiti Sherry , Lawres Lauren , Cardone Rebecca , Kibbey Richard , Krishnaswamy Smita , Muzumdar Mandar D
Published: January 2024

Epidemiologic studies have shown that obesity is a risk factor for more than a dozen cancers, including pancreatic ductal adenocarcinoma (PDAC). As PDAC is one of the leading causes of cancer-related death and obesity rates are rapidly increasing worldwide, it is imperative that we understand the mechanisms by which obesity promotes PDAC progression. To address this gap in knowledge, our lab combined a well-established genetic model for obesity (O: Lepob/ob) with a Kras-driven pancreatic cancer model (KC: Pdx1-Cre; KrasLSL-G12D/+) that mimics human PDAC progression. Resultant KCO mice had a drastic increase in tumor development compared to their lean counterparts, whereas induced weight loss intercepted tumor progression, showing that the tumor-promoting effects of obesity can be reversed. Bulk RNA sequencing of tumors from these mice revealed aberrantly increased expression of multiple peptide hormones expressed in insulin-producing beta (b) cells of pancreatic islets, including cholecystokinin (CCK) and amylin (Iapp). Strikingly, transgenic expression of CCK and Iapp in b-cells was sufficient to promote Kras-driven pancreatic tumorigenesis in non-obese mice. Single-cell RNA sequencing of b cells showed a trajectory of changes in gene expression with increasing obesity from wildtype to high-fat diet-fed to Lepob/ob mice. Further in silico analysis combining archetypal (AANet) with pseudotime (TrajectoryNet) methods demonstrated that b cells misexpressing hormones (CCK and Iapp) arose from a specific subtype of preexisting b cells, specifically virgin b cells, a rare, immature, regenerative population. Preliminary in vivo lineage tracing analyses in MIP-CreERT;LSL-TdTomato;Lepob/ob mice support these findings. Finally, we investigated the transcriptional networks that lead to aberrant CCK and Iapp expression in b cells. By combining Grainger causality analysis with known gene-gene relationships in the Transcriptional Regulatory Relationship Unraveled by Sentence-based Text mining (TRRUST) v2 database, we discovered a stress-induced mitogen-activated protein kinase (MAPK8)-cJun pathway that promotes pro-tumorigenic hormone expression in b cells. These combined experimental and computational methods nominate novel b cell targets to subvert the development of PDAC, an exocrine tumor.

Citation Format: Cathy C. Garcia, Aarthi Venkat, Alex Tong, Sherry Agabiti, Lauren Lawres, Rebecca Cardone, Richard Kibbey, Smita Krishnaswamy, Mandar D Muzumdar. Pancreatic beta cell stress pathways drive pancreatic ductal adenocarcinoma development in obesity [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 B049.
Abstract B051: Investigating lipid homeostasis in pancreatic ductal adenocarcinoma under tumor-like stress
Authors: Han Xu , Burrows Michelle , Kim Laura , Mesaros Clementina , Tobias John , Keith Brian , Simon Celeste
Published: January 2024

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a 5-year survival rate of only 10%. One hallmark of PDAC’s tumor microenvironment is dense desmoplasia, due to abnormal accumulation of extracellular matrix and proliferative fibroblasts. During tumorigenesis, pancreatic stellate cells (PSCs) obtain myofibroblast-like signatures and contribute to the fibrotic environment of PDAC. Desmoplasia-induced hypovascularity severely limits oxygen and nutrient delivery. Our laboratory revealed that hypoxia is prominent even at the pancreatic intraepithelial neoplasia (PanIN) stage, a dominant precursor lesion of PDAC. Hypoxia inhibits oxygen-dependent metabolic processes. For example, unsaturated lipid biosynthesis relies on oxygen to maintain desaturase enzymatic activities. Hypoxia also stimulates lipid uptake. Previous studies demonstrated that RAS-transformed cells preferentially import unsaturated lysophosphatidylcholines (LPCs) from culture media. However, in a nutrient-limited fibrotic environment, the potential source of lipids is unclear. Interestingly, a lipidomic study of a PSC secretome displayed a significant amount of LPCs, suggesting that PSCs are the potential lipid source in the PDAC microenvironment. Our data indicate that exogenous unsaturated lipids sustain PDAC cell viability under hypoxia and nutrient deficiency. Moreover, we find PDAC cell survival under tumor-like stress is enhanced in PSC conditioned medium, but not in delipidated conditioned medium. Strikingly, we show that cancer-associated fibroblasts do not experience hypoxic stress as much as adjacent malignant epithelium cells in vivo, suggesting cancer-associated fibroblasts provide unsaturated (and other) lipids to PDAC cells. Based on our lipidomic mass spectra of PSC conditioned medium, we determined that LPCs are actively utilized by PDAC cells for phosphatidylcholine and triglyceride regeneration. By inhibiting LPC to PC conversion with the drug TSI-01, which targets LPCAT (acyltransferase), unsaturated LPC no longer sustains cancer cell survival and ER stress is reactivated. In this study, we demonstrated metabolic crosstalk between fibroblasts and PDAC cells under stress conditions. Our findings revealed a novel metabolic inhibitor to disrupt the metabolic crosstalk between PSC and pancreatic cancer cells which might benefit developing combinatorial therapy of PDAC.

Citation Format: Xu Han, Michelle Burrows, Laura Kim, Clementina Mesaros, John Tobias, Brian Keith, Celeste Simon. Investigating lipid homeostasis in pancreatic ductal adenocarcinoma under tumor-like stress [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 B051.
Abstract B064: Redefining phenotypic heterogeneity of pancreatic ductal adenocarcinoma: A bottom-up approach
Authors: Hilmi Marc , Delecourt Flore , Rafenne Jerôme , Bourega Taib , Dusetti Nelson , Iovanna Juan , Blum Yuna , Richard Magali , Neuzillet Cindy , Couvelard Anne , De Mestier Louis , Rebours Vinciane , Nicolle Rémy , Cros Jérôme
Published: January 2024

Background: Pancreatic ductal adenocarcinoma (PDAC) tumor inter-patient heterogeneity has been well described with two major subtypes (classical and basal-like). An important intra-patient heterogeneity has not yet been extensively studied due to the lack of standardized and easily accessible high throughput methods compatible with clinical routine. Material and Methods: To build an immunohistochemical (IHC) tool capable of differentiating classical and basal-like tumors, relevant antibodies were selected using a multi-step process. The successive stages of an in-silico selection from the bulk transcriptome of 309 PDACs and 30 patient-derived xenografts followed by the validation on tissue microarrays in a cohort of PDAC (n=50) were conducted. We used our final IHC panel on whole slides of serial histology sections in 95 resected PDACs. After image registration, we performed a tile-based-analysis in tumoral and pre-neoplastic areas and a k-means clustering to identify relevant marker combinations. Results: Our final selection of the IHC panel included GATA6, CLN18, TFF1, MUC16, S100A2, KRT17, PanBasal. Among the 44,042 tumor tiles analyzed, 4 different phenotypes were identified: 1 classical (CLN18+, TFF1+, GATA6+), 1 intermediate (KRT17+) and 2 basal-like (MUC16+ vs S100A2+) with specific biological properties. A minor basal contingent drastically reduced overall survival (OS) in the multivariate analysis (HR=2.36, p=0.01). In contrast, the presence of the intermediate and classical clusters did not impact OS. However, among patients with PDAC expressing the classical cluster, a tremendous difference of OS was observed according to the co-occurence of a basal cluster (HR=0.34, p=0.003). These prognostic associations have been validated in an independent cohort of 150 patients with PDAC on tissue microarrays. By analyzing preneoplastic lesions, we showed the evolution process of pancreatic carcinogenesis going from a classical toward a basal through an early intermediate phenotype. Conclusion: Our restricted panel of antibodies could help pathologists to predict the high degree of PDAC heterogeneity, ultimately leading to an improvement of patient management.

Citation Format: Marc Hilmi, Flore Delecourt, Jerôme Rafenne, Taib Bourega, Nelson Dusetti, Juan Iovanna, Yuna Blum, Magali Richard, Cindy Neuzillet, Anne Couvelard, Louis De Mestier, Vinciane Rebours, Rémy Nicolle, Jérôme Cros. Redefining phenotypic heterogeneity of pancreatic ductal adenocarcinoma: A bottom-up approach [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 B064.
Abstract B069: Double-stranded RNA derived from tandem repeat sequences induces mesenchymal transition in pancreatic cancer cells by regulating alternative splicing
Authors: Kishikawa Takahiro , Iwata Takuma , Otsuka Motoyuki , Fujishiro Mitsuhiro
Published: January 2024

Human satellite II (HSATII) composed of tandem repeats in pericentromeric regions in the human genome is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of the repetitive elements in cancer tissues can facilitate incidental double-stranded RNA formation. However, it remains controversial whether double-stranded RNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the formation of double-stranded HSATII (dsHSATII) RNA and explored its molecular functions. The overexpression of dsHSATII RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, STRBP, which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. Depletion of STRBP induced the mesenchymal transition and enhanced the invasiveness. Conversely, the epithelial–mesenchymal transition (EMT) of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with the EMT events, which was interfered by the overexpression of dsHSATII RNA. Finally, we confirmed that isoform switching of CLSTN1, one of the target genes of STRBP-associated alternative splicing, induced EMT-like morphological changes. These findings reveal a novel cell-autonomous function of dsHSATII RNA in enhancing malignant potential, providing a better understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors.

Citation Format: Takahiro Kishikawa, Takuma Iwata, Motoyuki Otsuka, Mitsuhiro Fujishiro. Double-stranded RNA derived from tandem repeat sequences induces mesenchymal transition in pancreatic cancer cells by regulating alternative splicing [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 B069.
Abstract B104: Epigenetic small molecule library screen to discover compounds that inhibit and reverse pancreatic acinar to ductal metaplasia
Authors: Perkins Corey M. , Atanasova Kalina R. , Ratnayake Ranjala , Jiang Jinmai , Chen Qi-Yin , Schmittgen Thomas D. , Luesch Hendrik
Published: January 2024

Pancreatic ductal adenocarcinoma (PDAC) development is believed to be preluded by the event of acinar to ductal metaplasia (ADM), the transdifferentiation of acinar cells into a duct-like state. It is widely accepted that either acinar or ductal cells bearing mutant Kras drive the development of PDAC. We have previously shown that we can inhibit and reverse ADM in a 3D organoid assay using primary pancreatic tissue from p48Cre/+LSL-KRASG12D/+ mice using the histone deacetylase inhibitor (HDACi), Trichostatin A (Da Silva, 2022). We further employed our 3D organoid model to perform a phenotypic and molecular screening of 144 epigenetic compounds. We identified class I HDACi, FK228, and the histone methyltransferase inhibitor, Chaetocin, as the top hits of reversing ADM without inducing cytotoxicity as assessed by calcein AM staining. Largazole homodomer, a novel HDACi derived from marine cyanobacteria, was also identified as both an inhibitor and reversal agent of ADM. These three compounds demonstrated phenotypic and molecular reversal of duct-like structures back into their acinar state, in addition to relative mRNA upregulation of acinar markers (Amy2a, Cela1, Cpa2) and downregulation of ductal markers (Krt19, Krt7, Sox9). Bulk-RNA sequencing combined with pathway analysis demonstrated pronounced inhibition of cancer signaling pathways during ADM reversal, such as TGFβ1, Vegf, TNF, IL-6, and STAT3. Moreover, RNA sequencing showed significant upregulation and downregulation of acinar and ductal/PDAC markers, respectively. Thus, a novel phenotypic small molecular screen based on organoid morphology was developed to discover compounds that inhibit and reverse pancreatic ADM in a mouse model with mutant Kras.

Citation Format: Corey M. Perkins, Kalina R. Atanasova, Ranjala Ratnayake, Jinmai Jiang, Qi-Yin Chen, Thomas D. Schmittgen, Hendrik Luesch. Epigenetic small molecule library screen to discover compounds that inhibit and reverse pancreatic acinar to ductal metaplasia [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 B104.
Abstract B109: Reinforcing risk prediction for intraductal papillary mucinous neoplasms of the pancreas with AI-optimized nucleotide-to-amino acid analyses
Authors: Nguyen Nam , Teer Jamie K. , Park Margaret A. , McDonald Patricia , Fleming Jason B. , Permuth Jennifer B. , Chen Kwang-Cheng , Karolak Aleksandra
Published: January 2024

Intraductal papillary mucinous neoplasms (IPMNs) often precede invasive pancreatic ductal adenocarcinoma, with each IPMN tumor carrying a 10-25% risk of progressing to cancer within 10 years of detection. The objective of this work is to employ AI-augmented analyses of single mutations occurring at specific tri-nucleotide sequences or codons and identify markers associated with the progression of cysts to pancreatic cancer. Despite being precursors of pancreatic cancer there is a lack of validated intervention targets for precision prevention for IPMNs. Currently cancer prevention for patients with IPMN is centered on surgery using a risk-tailored approach; the clinical ability to stratify risk of cancer progression of individual IPMN tumors is poor and essentially no effective non-surgical interventions exist. To advance the precision cancer prevention for IPMNs we develop an intelligent, machine learning (ML) derived framework addressing the challenge of IPMN stratification from samples with limited data space, e.g., somatic mutations. Our study incorporates the text-based statistical and ML analyses of the mutational profiles of patients’ genomes followed by an integration of codon sequence derived mutational features for biomarkers validation. Given the potential of genome spatial organization to understand mutagenesis and genomic instability, we also map the DNA sequence information onto the local spatial genome organization to elucidate intrinsic features and molecular mechanisms that potentially lead to mutations. Results of this study show that information extracted from mutational profiles with the proposed methods significantly improves performance of the ML models in stratification of IPMNs and biomarker identification. We also present how utilization of these ML tools for mutational data, spatial genome organization, and functional analyses from tumor samples, can aid understanding of the underlying mechanisms of mutations in IPMNs and pancreatic cancer.

Citation Format: Nam Nguyen, Jamie K. Teer, Margaret A. Park, Patricia McDonald, Jason B. Fleming, Jennifer B. Permuth, Kwang-Cheng Chen, Aleksandra Karolak. Reinforcing risk prediction for intraductal papillary mucinous neoplasms of the pancreas with AI-optimized nucleotide-to-amino acid analyses [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 B109.