Netrin G1 Ligand is a new stromal immunomodulator that promotes pancreatic cancer.

Understanding pancreatic cancer biology is fundamental for identifying new targets and for developing more effective therapies. In particular, the contribution of the stromal microenvironment to pancreatic cancer tumorigenesis requires further exploration. Here, we report the stromal roles of the synaptic protein Netrin G1 Ligand (NGL-1) in pancreatic cancer, uncovering its pro-tumor functions in cancer-associated fibroblasts and in immune cells. We observed that the stromal expression of NGL-1 inversely correlated with patients' overall survival. Moreover, germline knockout (KO) mice for NGL-1 presented decreased tumor burden, with a microenvironment that is less supportive of tumor growth. Of note, tumors from NGL-1 KO mice produced less immunosuppressive cytokines and displayed an increased percentage of CD8 + T cells than those from control mice, while preserving the physical structure of the tumor microenvironment. These effects were shown to be mediated by NGL-1 in both immune cells and in the local stroma, in a TGF-ß-dependent manner. While myeloid cells lacking NGL-1 decreased the production of immunosuppressive cytokines, NGL-1 KO T cells showed increased proliferation rates and overall polyfunctionality compared to control T cells. CAFs lacking NGL-1 were less immunosuppressive than controls, with overall decreased production of pro-tumor cytokines and compromised ability to inhibit CD8 + T cells activation. Mechanistically, these CAFs downregulated components of the TGF-ß pathway, AP-1 and NFAT transcription factor families, resulting in a less tumor-supportive phenotype. Finally, targeting NGL-1 genetically or using a functionally antagonistic small peptide phenocopied the effects of chemotherapy, while modulating the immunosuppressive tumor microenvironment (TME), rather than eliminating it. We propose NGL-1 as a new local stroma and immunomodulatory molecule, with pro-tumor roles in pancreatic cancer. Statement of Significance: Here we uncovered the pro-tumor roles of the synaptic protein NGL-1 in the tumor microenvironment of pancreatic cancer, defining a new target that simultaneously modulates tumor cell, fibroblast, and immune cell functions. This study reports a new pathway where NGL-1 controls TGF-ß, AP-1 transcription factor members and NFAT1, modulating the immunosuppressive microenvironment in pancreatic cancer. Our findings highlight NGL-1 as a new stromal immunomodulator in pancreatic cancer.

Tuft cells transdifferentiate to neural-like progenitor cells in the progression of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is partly initiated through the transdifferentiation of acinar cells to metaplastic ducts that act as precursors of neoplasia and cancer. Tuft cells are solitary chemosensory cells not found in the normal pancreas but arise in metaplasia and neoplasia, diminishing as neoplastic lesions progress to carcinoma. Metaplastic tuft cells (mTCs) function to suppress tumor progression through communication with the tumor microenvironment, but their fate during progression is unknown. To determine the fate of mTCs during PDA progression, we have created a lineage tracing model that uses a tamoxifen-inducible tuft-cell specific Pou2f3CreERT/+ driver to induce transgene expression, including the lineage tracer tdTomato or the oncogene Myc. mTC lineage trace models of pancreatic neoplasia and carcinoma were used to follow mTC fate. We found that mTCs, in the carcinoma model, transdifferentiate into neural-like progenitor cells (NRPs), a cell type associated with poor survival in PDA patients. Using conditional knock-out and overexpression systems, we found that Myc activity in mTCs is necessary and sufficient to induce this Tuft-to-Neuroendocrine-Transition (TNT).

ROR2 regulates cellular plasticity in pancreatic neoplasia and adenocarcinoma.

Cellular plasticity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) starting from the conversion of normal cells into precancerous lesions to the progression of carcinoma subtypes associated with aggressiveness and therapeutic response. We discovered that normal acinar cell differentiation, maintained by the transcription factor Pdx1, suppresses a broad gastric cell identity that is maintained in metaplasia, neoplasia, and the classical subtype of PDAC in mouse and human. We have identified the receptor tyrosine kinase Ror2 as marker of a gastric metaplasia (SPEM)-like identity in the pancreas. Ablation of Ror2 in a mouse model of pancreatic tumorigenesis promoted a switch to a gastric pit cell identity that largely persisted through progression to the classical subtype of PDAC. In both human and mouse pancreatic cancer, ROR2 activity continued to antagonize the gastric pit cell identity, strongly promoting an epithelial to mesenchymal transition, conferring resistance to KRAS inhibition, and vulnerability to AKT inhibition.

Notch Signaling Regulates Immunosuppressive Tumor-Associated Macrophage Function in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) continues to have a dismal prognosis. The poor survival of patients with PDA has been attributed to a high rate of early metastasis and low efficacy of current therapies, which partly result from its complex immunosuppressive tumor microenvironment. Previous studies from our group and others have shown that tumor-associated macrophages (TAM) are instrumental in maintaining immunosuppression in PDA. Here, we explored the role of Notch signaling, a key regulator of immune response, within the PDA microenvironment. We identified Notch pathway components in multiple immune cell types within human and mouse pancreatic cancer. TAMs, the most abundant immune cell population in the tumor microenvironment, expressed high levels of Notch receptors, with cognate ligands such as JAG1 expressed on tumor epithelial cells, endothelial cells, and fibroblasts. TAMs with activated Notch signaling expressed higher levels of immunosuppressive mediators, suggesting that Notch signaling plays a role in macrophage polarization within the PDA microenvironment. Genetic inhibition of Notch in myeloid cells led to reduced tumor size and decreased macrophage infiltration in an orthotopic PDA model. Combination of pharmacologic Notch inhibition with PD-1 blockade resulted in increased cytotoxic T-cell infiltration, tumor cell apoptosis, and smaller tumor size. Our work implicates macrophage Notch signaling in the establishment of immunosuppression and indicates that targeting the Notch pathway may improve the efficacy of immune-based therapies in patients with PDA.

Modeling Molecular Pathogenesis of Idiopathic Pulmonary Fibrosis-Associated Lung Cancer in Mice.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive, often fatal loss of lung function due to overactive collagen production and tissue scarring. Patients with IPF have a sevenfold-increased risk of developing lung cancer. The COVID-19 pandemic has increased the number of patients with lung diseases, and infection can worsen prognoses for those with chronic lung diseases and disease-associated cancer. Understanding the molecular pathogenesis of IPF-associated lung cancer is imperative for identifying diagnostic biomarkers and targeted therapies that will facilitate prevention of IPF and progression to lung cancer. To understand how IPF-associated fibroblast activation, matrix remodeling, epithelial-to-mesenchymal transition (EMT), and immune modulation influences lung cancer predisposition, we developed a mouse model to recapitulate the molecular pathogenesis of pulmonary fibrosis-associated lung cancer using the bleomycin and Lewis lung carcinoma models. We demonstrate that development of pulmonary fibrosis-associated lung cancer is likely linked to increased abundance of tumor-associated macrophages and a unique gene signature that supports an immune-suppressive microenvironment through secreted factors. Not surprisingly, preexisting fibrosis provides a pre-metastatic niche and results in augmented tumor growth, and tumors associated with bleomycin-induced fibrosis are characterized by a dramatic loss of cytokeratin expression, indicative of EMT. IMPLICATIONS: This characterization of tumors associated with lung diseases provides new therapeutic targets that may aid in the development of treatment paradigms for lung cancer patients with preexisting pulmonary diseases.

Multi-omics analysis of metastatic pancreatic cancer reveals an immunosuppressive landscape.

Immune cell populations play key functional roles in tumor growth and metastasis, and multi-omic approaches can map cellular interactions to identify targets for highly immunosuppressive and treatment resistant cancers. In this issue of Med, Zhang et al.1 investigate the immune cell landscape of advanced pancreatic cancer to understand the mechanisms underlying liver metastasis.

KRT17High/CXCL8+ tumor cells display both classical and basal features and regulate myeloid infiltration in the pancreatic cancer microenvironment.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is generally divided in two subtypes, classical and basal. Recently, single cell RNA sequencing has uncovered the co-existence of basal and classical cancer cells, as well as intermediary cancer cells, in individual tumors. The latter remains poorly understood; here, we sought to characterize them using a multimodal approach. EXPERIMENTAL DESIGN: We performed subtyping on a single cell RNA sequencing dataset containing 18 human PDAC samples to identify multiple intermediary subtypes. We generated patient-derived PDAC organoids for functional studies. We compared single cell profiling of matched blood and tumor samples to measure changes in the local and systemic immune microenvironment. We then leveraged longitudinally patient-matched blood to follow individual patients over the course of chemotherapy. RESULTS: We identified a cluster of KRT17-high intermediary cancer cells that uniquely express high levels of CXCL8 and other cytokines. The proportion of KRT17High/CXCL8+ cells in patient tumors correlated with intra-tumoral myeloid abundance, and, interestingly, high pro-tumor peripheral blood granulocytes, implicating local and systemic roles. Patient-derived organoids maintained KRT17High/CXCL8+cells and induced myeloid cell migration in an CXCL8-dependent manner. In our longitudinal studies, plasma CXCL8 decreased following chemotherapy in responsive patients, while CXCL8 persistence portended worse prognosis. CONCLUSIONS: Through single cell analysis of PDAC samples we identified KRT17High/CXCL8+ cancer cells as an intermediary subtype, marked by a unique cytokine profile and capable of influencing myeloid cells in the tumor microenvironment and systemically. The abundance of this cell population should be considered for patient stratification in precision immunotherapy.

Lorazepam Stimulates IL6 Production and Is Associated with Poor Survival Outcomes in Pancreatic Cancer.

PURPOSE: This research investigates the association between benzodiazepines (BZD) and cancer patient survival outcomes, the pancreatic cancer tumor microenvironment, and cancer-associated fibroblast (CAF) signaling. EXPERIMENTAL DESIGN: Multivariate Cox regression modeling was used to retrospectively measure associations between Roswell Park cancer patient survival outcomes and BZD prescription records. IHC, H&E, Masson's trichrome, RNAscope, and RNA sequencing were used to evaluate the impact of lorazepam (LOR) on the murine PDAC tumor microenvironment. ELISA and qPCR were used to determine the impact of BZDs on IL6 expression or secretion by human-immortalized pancreatic CAFs. PRESTO-Tango assays, reanalysis of PDAC single-cell sequencing/TCGA data sets, and GPR68 CRISPRi knockdown CAFs were used to determine the impact of BZDs on GPR68 signaling. RESULTS: LOR is associated with worse progression-free survival (PFS), whereas alprazolam (ALP) is associated with improved PFS, in pancreatic cancer patients receiving chemotherapy. LOR promotes desmoplasia (fibrosis and extracellular matrix protein deposition), inflammatory signaling, and ischemic necrosis. GPR68 is preferentially expressed on human PDAC CAFs, and n-unsubstituted BZDs, such as LOR, significantly increase IL6 expression and secretion in CAFs in a pH and GPR68-dependent manner. Conversely, ALP and other GPR68 n-substituted BZDs decrease IL6 in human CAFs in a pH and GPR68-independent manner. Across many cancer types, LOR is associated with worse survival outcomes relative to ALP and patients not receiving BZDs. CONCLUSIONS: We demonstrate that LOR stimulates fibrosis and inflammatory signaling, promotes desmoplasia and ischemic necrosis, and is associated with decreased pancreatic cancer patient survival.

Notch signaling regulates immunosuppressive tumor-associated macrophage function in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) continues to have a dismal prognosis. The poor survival of patients with PDA has been attributed to a high rate of early metastasis and low efficacy of current therapies, which partly result from its complex immunosuppressive tumor microenvironment. Previous studies from our group and others have shown that tumor-associated macrophages (TAMs) are instrumental in maintaining immunosuppression in PDA. Here, we explored the role of Notch signaling, a key regulator of immune response, within the PDA microenvironment. We identified Notch pathway components in multiple immune cell types within human and mouse pancreatic cancer. TAMs, the most abundant immune cell population in the tumor microenvironment, express high levels of Notch receptors with cognate ligands such as JAG1 expressed on tumor epithelial cells, endothelial cells and fibroblasts. TAMs with activated Notch signaling expressed higher levels of immunosuppressive mediators including arginase 1 (Arg1) suggesting that Notch signaling plays a role in macrophage polarization within the PDA microenvironment. Combination of Notch inhibition with PD-1 blockade resulted in increased cytotoxic T cell infiltration, tumor cell apoptosis, and smaller tumor size. Our work implicates macrophage Notch signaling in the establishment of immunosuppression and indicates that targeting the Notch pathway may improve the efficacy of immune-based therapies in PDA patients.

Differential integrated stress response and asparagine production drive symbiosis and therapy resistance of pancreatic adenocarcinoma cells.

The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor. However, it remains unclear if this diversity extends to metabolic programming. Here, using metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses among neoplastic populations within individual human and mouse tumors. Furthermore, these populations are poised for metabolic cross-talk, and in examining this, we find an unexpected role for asparagine supporting proliferation during limited respiration. Constitutive GCN2 activation permits ATF4 signaling in one subtype, driving excess asparagine production. Asparagine release provides resistance during impaired respiration, enabling symbiosis. Functionally, availability of exogenous asparagine during limited respiration indirectly supports maintenance of aspartate pools, a rate-limiting biosynthetic precursor. Conversely, depletion of extracellular asparagine with PEG-asparaginase sensitizes tumors to mitochondrial targeting with phenformin.

Metabolic requirement for GOT2 in pancreatic cancer depends on environmental context.

Mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2) is part of the malate-aspartate shuttle, a mechanism by which cells transfer reducing equivalents from the cytosol to the mitochondria. GOT2 is a key component of mutant KRAS (KRAS*)-mediated rewiring of glutamine metabolism in pancreatic ductal adenocarcinoma (PDA). Here, we demonstrate that the loss of GOT2 disturbs redox homeostasis and halts proliferation of PDA cells in vitro. GOT2 knockdown (KD) in PDA cell lines in vitro induced NADH accumulation, decreased Asp and α-ketoglutarate (αKG) production, stalled glycolysis, disrupted the TCA cycle, and impaired proliferation. Oxidizing NADH through chemical or genetic means resolved the redox imbalance induced by GOT2 KD, permitting sustained proliferation. Despite a strong in vitro inhibitory phenotype, loss of GOT2 had no effect on tumor growth in xenograft PDA or autochthonous mouse models. We show that cancer-associated fibroblasts (CAFs), a major component of the pancreatic tumor microenvironment (TME), release the redox active metabolite pyruvate, and culturing GOT2 KD cells in CAF conditioned media (CM) rescued proliferation in vitro. Furthermore, blocking pyruvate import or pyruvate-to-lactate reduction prevented rescue of GOT2 KD in vitro by exogenous pyruvate or CAF CM. However, these interventions failed to sensitize xenografts to GOT2 KD in vivo, demonstrating the remarkable plasticity and differential metabolism deployed by PDA cells in vitro and in vivo. This emphasizes how the environmental context of distinct pre-clinical models impacts both cell-intrinsic metabolic rewiring and metabolic crosstalk with the TME.

Combinatorial Gli activity directs immune infiltration and tumor growth in pancreatic cancer.

Proper Hedgehog (HH) signaling is essential for embryonic development, while aberrant HH signaling drives pediatric and adult cancers. HH signaling is frequently dysregulated in pancreatic cancer, yet its role remains controversial, with both tumor-promoting and tumor-restraining functions reported. Notably, the GLI family of HH transcription factors (GLI1, GLI2, GLI3), remain largely unexplored in pancreatic cancer. We therefore investigated the individual and combined contributions of GLI1-3 to pancreatic cancer progression. At pre-cancerous stages, fibroblast-specific Gli2/Gli3 deletion decreases immunosuppressive macrophage infiltration and promotes T cell infiltration. Strikingly, combined loss of Gli1/Gli2/Gli3 promotes macrophage infiltration, indicating that subtle changes in Gli expression differentially regulate immune infiltration. In invasive tumors, Gli2/Gli3 KO fibroblasts exclude immunosuppressive myeloid cells and suppress tumor growth by recruiting natural killer cells. Finally, we demonstrate that fibroblasts directly regulate macrophage and T cell migration through the expression of Gli-dependent cytokines. Thus, the coordinated activity of GLI1-3 directs the fibroinflammatory response throughout pancreatic cancer progression.

Extrinsic KRAS Signaling Shapes the Pancreatic Microenvironment Through Fibroblast Reprogramming.

BACKGROUND & AIMS: Oncogenic Kirsten Rat Sarcoma virus (KRAS) is the hallmark mutation of human pancreatic cancer and a driver of tumorigenesis in genetically engineered mouse models of the disease. Although the tumor cell-intrinsic effects of oncogenic Kras expression have been widely studied, its role in regulating the extensive pancreatic tumor microenvironment is less understood. METHODS: Using a genetically engineered mouse model of inducible and reversible oncogenic Kras expression and a combination of approaches that include mass cytometry and single-cell RNA sequencing we studied the effect of oncogenic KRAS in the tumor microenvironment. RESULTS: We have discovered that non-cell autonomous (ie, extrinsic) oncogenic KRAS signaling reprograms pancreatic fibroblasts, activating an inflammatory gene expression program. As a result, fibroblasts become a hub of extracellular signaling, and the main source of cytokines mediating the polarization of protumorigenic macrophages while also preventing tissue repair. CONCLUSIONS: Our study provides fundamental knowledge on the mechanisms underlying the formation of the fibroinflammatory stroma in pancreatic cancer and highlights stromal pathways with the potential to be exploited therapeutically.

Multiomic characterization of pancreatic cancer-associated macrophage polarization reveals deregulated metabolic programs driven by the GM-CSF-PI3K pathway.

The pancreatic ductal adenocarcinoma microenvironment is composed of a variety of cell types and marked by extensive fibrosis and inflammation. Tumor-associated macrophages (TAMs) are abundant, and they are important mediators of disease progression and invasion. TAMs are polarized in situ to a tumor promoting and immunosuppressive phenotype via cytokine signaling and metabolic crosstalk from malignant epithelial cells and other components of the tumor microenvironment. However, the specific distinguishing features and functions of TAMs remain poorly defined. Here, we generated tumor-educated macrophages (TEMs) in vitro and performed detailed, multiomic characterization (i.e., transcriptomics, proteomics, metabolomics). Our results reveal unique genetic and metabolic signatures of TEMs, the veracity of which were queried against our in-house single-cell RNA sequencing dataset of human pancreatic tumors. This analysis identified expression of novel, metabolic TEM markers in human pancreatic TAMs, including ARG1, ACLY, and TXNIP. We then utilized our TEM model system to study the role of mutant Kras signaling in cancer cells on TEM polarization. This revealed an important role for granulocyte-macrophage colony-stimulating factor (GM-CSF) and lactate on TEM polarization, molecules released from cancer cells in a mutant Kras-dependent manner. Lastly, we demonstrate that GM-CSF dysregulates TEM gene expression and metabolism through PI3K-AKT pathway signaling. Collectively, our results define new markers and programs to classify pancreatic TAMs, how these are engaged by cancer cells, and the precise signaling pathways mediating polarization.

Disruption of Her2-Induced PD-L1 Inhibits Tumor Cell Immune Evasion in Patient-Derived Gastric Cancer Organoids.

(1) Background: The expression of programmed death-ligand 1 (PD-L1), which interacts with programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTLs), enables tumors to escape immunosurveillance. The PD-1/PD-L1 interaction results in the inhibition of CTL proliferation, and effector function, thus promoting tumor cell evasion from immunosurveillance and cancer persistence. Despite 40% of gastric cancer patients exhibiting PD-L1 expression, only a small subset of patients responds to immunotherapy. Human epidermal growth factor receptor2 (HER2) is one of the critical regulators of several solid tumors, including metastatic gastric cancer. Although half of PD-L1-positive gastric tumors co-express HER2, crosstalk between HER2 and PD-1/PD-L1 in gastric cancer remains undetermined. (2) Methods: Human gastric cancer organoids (huTGOs) were generated from biopsied or resected tissues and co-cultured with CTLs and myeloid-derived suppressor cells (MDSCs). Digital Spatial Profiling (DSP) was performed on FFPE tissue microarrays of numerous gastric cancer patients to examine the protein expression of immune markers. (3) Results: Knockdown of HER2 in PD-L1/HER2-positive huTGOs led to a concomitant decrease in PD-L1 expression. Similarly, in huTGOs/immune cell co-cultures, PD-L1 expression decreased in huTGOs and was correlated with an increase in CTL proliferation which enhanced huTGO death. Treatment with Nivolumab exhibited similar effects. However, a combinatorial treatment with Mubritinib and Nivolumab was unable to inhibit HER2 expression in co-cultures containing MDSCs. (4) Conclusions: Our study suggested that co-expression of HER2 and PD-L1 may contribute to tumor cell immune evasion. In addition, autologous organoid/immune cell co-cultures can be exploited to effectively screen responses to a combination of anti-HER2 and immunotherapy to tailor treatment for gastric cancer patients.

Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity.

Fibroblasts display extensive transcriptional heterogeneity, yet functional annotation and characterization of their heterocellular relationships remains incomplete. Using mass cytometry, we chart the stromal composition of 18 murine tissues and 5 spontaneous tumor models, with an emphasis on mesenchymal phenotypes. This analysis reveals extensive stromal heterogeneity across tissues and tumors, and identifies coordinated relationships between mesenchymal and immune cell subsets in pancreatic ductal adenocarcinoma. Expression of CD105 demarks two stable and functionally distinct pancreatic fibroblast lineages, which are also identified in murine and human healthy tissues and tumors. Whereas CD105-positive pancreatic fibroblasts are permissive for tumor growth in vivo, CD105-negative fibroblasts are highly tumor suppressive. This restrictive effect is entirely dependent on functional adaptive immunity. Collectively, these results reveal two functionally distinct pancreatic fibroblast lineages and highlight the importance of mesenchymal and immune cell interactions in restricting tumor growth.

Hedgehog transcriptional effector GLI mediates mTOR-Induced PD-L1 expression in gastric cancer organoids.

Tumors evade immune surveillance by expressing Programmed Death-Ligand 1 (PD-L1), subsequently inhibiting CD8+ cytotoxic T lymphocyte function. Response of gastric cancer to immunotherapy is relatively low. Our laboratory has reported that Helicobacter pylori-induced PD-L1 expression within the gastric epithelium is mediated by the Hedgehog (Hh) signaling pathway. The PI3K/AKT/mTOR pathway is activated in gastric cancer and may have immunomodulatory potential. We hypothesize that Hh signaling mediates mTOR-induced PD-L1 expression. Patient-derived organoids (PDOs) were generated from gastric biopsies and resected tumor tissues. Autologous organoid/immune cell co-cultures were used to study the immunosuppressive function of MDSCs. NanoString Digital Spatial Profiling (DSP) of immune-related protein markers using FFPE slide-mounted tissues from gastric cancer patients was performed. DSP analysis showed infiltration of immunosuppressive MDSCs expressing Arg1, CD66b, VISTA and IDO1 within cancer tissues. Orthotopic transplantation of patient derived organoids (PDOs) resulted in the engraftment of organoids and the development of histology similar to that observed in the patient's tumor tissue. PDO/immune cell co-cultures revealed that PD-L1-expressing organoids were unresponsive to nivolumab in vitro in the presence of PMN-MDSCs. Depletion of PMN-MDSCs within these co-cultures sensitized the organoids to anti-PD-1/PD-L1-induced cancer cell death. Rapamycin decreased phosphorylated S6K, Gli2 and PD-L1 expression in PDO/immune cell co-cultures. Transcriptional regulation of PD-L1 by GLI1 and GLI2 was blocked by rapamycin. In conclusion, the PDO/immune cell co-cultures may be used to study immunosuppressive MDSC function within the gastric tumor microenvironment. The mTOR signaling pathway mediates GLI-induced PD-L1 expression in gastric cancer.

Apolipoprotein E Promotes Immune Suppression in Pancreatic Cancer through NF-κB-Mediated Production of CXCL1.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with few effective therapeutic options. PDAC is characterized by an extensive fibroinflammatory stroma that includes abundant infiltrating immune cells. Tumor-associated macrophages (TAM) are prevalent within the stroma and are key drivers of immunosuppression. TAMs in human and murine PDAC are characterized by elevated expression of apolipoprotein E (ApoE), an apolipoprotein that mediates cholesterol metabolism and has known roles in cardiovascular and Alzheimer's disease but no known role in PDAC. We report here that ApoE is also elevated in peripheral blood monocytes in PDAC patients, and plasma ApoE protein levels stratify patient survival. Orthotopic implantation of mouse PDAC cells into syngeneic wild-type or in ApoE-/- mice showed reduced tumor growth in ApoE-/- mice. Histologic and mass cytometric (CyTOF) analysis of these tumors showed an increase in CD8+ T cells in tumors in ApoE-/- mice. Mechanistically, ApoE induced pancreatic tumor cell expression of Cxcl1 and Cxcl5, known immunosuppressive factors, through LDL receptor and NF-κB signaling. Taken together, this study reveals a novel immunosuppressive role of ApoE in the PDAC microenvironment. SIGNIFICANCE: This study shows that elevated apolipoprotein E in PDAC mediates immune suppression and high serum apolipoprotein E levels correlate with poor patient survival.See related commentary by Sherman, p. 4186.

Loss of activating transcription factor 3 prevents KRAS-mediated pancreatic cancer.

The unfolded protein response (UPR) is activated in pancreatic pathologies and suggested as a target for therapeutic intervention. In this study, we examined activating transcription factor 3 (ATF3), a mediator of the UPR that promotes acinar-to-ductal metaplasia (ADM) in response to pancreatic injury. Since ADM is an initial step in the progression to pancreatic ductal adenocarcinoma (PDAC), we hypothesized that ATF3 is required for initiation and progression of PDAC. We generated mice carrying a germline mutation of Atf3 (Atf3-/-) combined with acinar-specific induction of oncogenic KRAS (Ptf1acreERT/+KrasG12D/+). Atf3-/- mice with (termed APK) and without KRASG12D were exposed to cerulein-induced pancreatitis. In response to recurrent pancreatitis, Atf3-/- mice showed decreased ADM and enhanced regeneration based on morphological and biochemical analysis. Similarly, an absence of ATF3 reduced spontaneous pancreatic intraepithelial neoplasia (PanIN) formation and PDAC in Ptf1acreERT/+KrasG12D/+ mice. In response to injury, KRASG12D bypassed the requirement for ATF3 with a dramatic loss in acinar tissue and PanIN formation observed regardless of ATF3 status. Compared to Ptf1acreERT/+KrasG12D/+ mice, APK mice exhibited a significant decrease in pancreatic and total body weight, did not progress through to PDAC, and showed altered pancreatic fibrosis and immune cell infiltration. These findings suggest a complex, multifaceted role for ATF3 in pancreatic cancer pathology.

Modeling pancreatic pathophysiology using genome editing of adult stem cell-derived and induced pluripotent stem cell (iPSC)-derived organoids.

In recent years, organoids have become a novel in vitro method to study gastrointestinal organ development, physiology, and disease. An organoid, in short, may be defined as a miniaturized organ that can be grown from adult stem cells in vitro and studied at the microscopic level. Organoids have been used in multitudes of different ways to study the physiology of different human diseases including gastrointestinal cancers such as pancreatic cancer. The development of genome editing based on the bacterial defense mechanism clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has emerged as a laboratory tool that provides the opportunity to study the effects of specific genetic changes on organ development, physiology, and disease. The CRISPR/Cas9 approach can be combined with organoid technology including the use of induced pluripotent stem cell (iPSC)-derived and tissue-derived organoids. The goal of this review is to provide highlights on the development of organoid technology, and the use of this culture system to study the pathophysiology of specific mutations in the development of pancreatic and gastric cancers.NEW & NOTEWORTHY The goal of this review is not only to provide highlights on the development of organoid technology but also to subsequently use this information to study the pathophysiology of those specific mutations in the formation of malignant pancreatic and gastric cancer.