Uridine-derived ribose fuels glucose-restricted pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease notoriously resistant to therapy1,2. This is mediated in part by a complex tumour microenvironment3, low vascularity4, and metabolic aberrations5,6. Although altered metabolism drives tumour progression, the spectrum of metabolites used as nutrients by PDA remains largely unknown. Here we identified uridine as a fuel for PDA in glucose-deprived conditions by assessing how more than 175 metabolites impacted metabolic activity in 21 pancreatic cell lines under nutrient restriction. Uridine utilization strongly correlated with the expression of uridine phosphorylase 1 (UPP1), which we demonstrate liberates uridine-derived ribose to fuel central carbon metabolism and thereby support redox balance, survival and proliferation in glucose-restricted PDA cells. In PDA, UPP1 is regulated by KRAS-MAPK signalling and is augmented by nutrient restriction. Consistently, tumours expressed high UPP1 compared with non-tumoural tissues, and UPP1 expression correlated with poor survival in cohorts of patients with PDA. Uridine is available in the tumour microenvironment, and we demonstrated that uridine-derived ribose is actively catabolized in tumours. Finally, UPP1 deletion restricted the ability of PDA cells to use uridine and blunted tumour growth in immunocompetent mouse models. Our data identify uridine utilization as an important compensatory metabolic process in nutrient-deprived PDA cells, suggesting a novel metabolic axis for PDA therapy.

Global Prevalence of Pancreatic Cystic Lesions in the General Population on Magnetic Resonance Imaging: A Systematic Review and Meta-analysis.

BACKGROUND & AIMS: Understanding the burden of pancreatic cystic lesions (PCLs) in the general population is important for clinicians and policymakers. In this systematic review, we sought to estimate the global prevalence of PCLs using magnetic resonance imaging (MRI) and to investigate factors that contribute to its variation. METHODS: We searched MEDLINE, EMBASE, and Cochrane Central, from database inception through February 2023. We included full-text articles that reported the prevalence of PCLs using MRI in the general population. A proportional meta-analysis was performed, and the prevalence of PCLs was pooled using a random-effects model. RESULTS: Fifteen studies with 65,607 subjects were identified. The pooled prevalence of PCLs was 16% (95% confidence interval [CI], 13%-18%; I2 = 99%), most of which were under 10 mm. Age-specific prevalence of PCLs increased from 9% (95% CI, 7%-12%) at 50 to 59 years, to 18% (95% CI, 14%-22%) at 60 to 69 years, 26% (95% CI, 20%-33%) at 70 to 79 years, and 38% at 80 years and above (95% CI, 25%-52%). There was no difference in prevalence between sexes. Subgroup analysis showed higher PCL prevalence when imaging findings were confirmed by independent radiologist(s) (25%; 95% CI, 16%-33%) than when chart review alone was used (5%; 95% CI, 4%-7%; P < .01). There was no independent association of PCL prevalence with geographic location (Europe, North America, or Asia), MRI indication (screening vs evaluation of non-pancreatic pathology), enrollment period, sample size, magnet strength (1.5 vs 3 tesla), and MRI sequence (magnetic resonance cholangiopancreatography vs no magnetic resonance cholangiopancreatography). CONCLUSION: In this systematic review, the global prevalence of PCLs using a highly sensitive noninvasive imaging modality ranged between 13% and 18%.

Effect of the number of passes on diagnostic performance of EUS-fine needle biopsy of solid pancreatic masses: A systematic review and meta-analysis.

BACKGROUND AND AIMS: The optimal number of passes to maximize the diagnostic ability of endoscopic ultrasound fine needle biopsy (EUS-FNB) of solid pancreatic masses (SPMs) is not well known. We conducted a systematic review to evaluate the impact of the incremental number of passes on diagnostic accuracy, tissue adequacy, and diagnostic yield for EUS-FNB of SPMs. METHODS: We searched MEDLINE, EMBASE, Scopus, and Cochrane Central for randomized controlled trials (RCTs) comparing per-pass diagnostic outcomes of FNB needles in patients with SPMs. Meta-analysis was conducted using random effects models. A separate analysis was performed on studies that used contemporary Franseen and fork-tip needles. RESULTS: Overall, 19 RCTs (N=3,552) were identified. For EUS-FNB of SPMs, three passes with any FNB needle outperformed two passes for accuracy (OR=1.58; 95%CI 1.20-2.09; I2=0%), adequacy (OR=1.97; 95%CI 1.30-2.83; I2=61%) and yield (OR=2.12; 95%CI 1.37-3.27; I2 14%). Adding a fourth or fifth pass resulted in no significant improvement in diagnostic parameters. When using contemporary FNB needles, adding a second to a single pass significantly improved accuracy (OR=1.80; 95%CI 1.23-2.63; I2=0%), adequacy (OR=2.19; 95% CI 1.65-2.90; I2=0%) and yield (OR=2.72; 95%CI 1.50-4.95; I2=0%). Adding a third pass to a second pass with contemporary needles improved adequacy (OR=2.96; 95%CI 1.97-4.46; I2=0%) but did not provide better diagnostic accuracy or yield. CONCLUSION: Two passes with Franseen or Fork-tip needles and three passes with any FNB needle suffice to provide optimal diagnostic performance for EUS-FNB of SPMs, without additional diagnostic benefits with more passes. Our results can inform future guidelines and quality benchmarks.

PI3K regulation of RAC1 is required for KRAS-induced pancreatic tumorigenesis in mice.

BACKGROUND & AIMS: New drug targets are urgently needed for the treatment of patients with pancreatic ductal adenocarcinoma (PDA). Nearly all PDAs contain oncogenic mutations in the KRAS gene. Pharmacological inhibition of KRAS has been unsuccessful, leading to a focus on downstream effectors that are more easily targeted with small molecule inhibitors. We investigated the contributions of phosphoinositide 3-kinase (PI3K) to KRAS-initiated tumorigenesis. METHODS: Tumorigenesis was measured in the Kras(G12D/+);Ptf1a(Cre/+) mouse model of PDA; these mice were crossed with mice with pancreas-specific disruption of genes encoding PI3K p110α (Pik3ca), p110ß (Pik3cb), or RAC1 (Rac1). Pancreatitis was induced with 5 daily intraperitoneal injections of cerulein. Pancreata and primary acinar cells were isolated; acinar cells were incubated with an inhibitor of p110α (PIK75) followed by a broad-spectrum PI3K inhibitor (GDC0941). PDA cell lines (NB490 and MiaPaCa2) were incubated with PIK75 followed by GDC0941. Tissues and cells were analyzed by histology, immunohistochemistry, quantitative reverse-transcription polymerase chain reaction, and immunofluorescence analyses for factors involved in the PI3K signaling pathway. We also examined human pancreas tissue microarrays for levels of p110α and other PI3K pathway components. RESULTS: Pancreas-specific disruption of Pik3ca or Rac1, but not Pik3cb, prevented the development of pancreatic tumors in Kras(G12D/+);Ptf1a(Cre/+) mice. Loss of transformation was independent of AKT regulation. Preneoplastic ductal metaplasia developed in mice lacking pancreatic p110α but regressed. Levels of activated and total RAC1 were higher in pancreatic tissues from Kras(G12D/+);Ptf1a(Cre/+) mice compared with controls. Loss of p110α reduced RAC1 activity and expression in these tissues. p110α was required for the up-regulation and activity of RAC guanine exchange factors during tumorigenesis. Levels of p110α and RAC1 were increased in human pancreatic intraepithelial neoplasias and PDAs compared with healthy pancreata. CONCLUSIONS: KRAS signaling, via p110α to activate RAC1, is required for transformation in Kras(G12D/+);Ptf1a(Cre/+) mice.

Endoscopic Ultrasound-Guided Ablation of Premalignant Pancreatic Cysts and Pancreatic Cancer.

Pancreatic cancer is on the rise and expected to become the second leading cause of cancer-related death by 2030. Up to a one-fifth of pancreatic cancers may arise from mucinous pancreatic cysts, which are frequently present in the general population. Currently, surgical resection is the only curative approach for pancreatic cancer and its cystic precursors. However, only a dismal proportion of patients are eligible for surgery. Therefore, novel treatment approaches to treat pancreatic cancer and precancerous pancreatic cysts are needed. Endoscopic ultrasound (EUS)-guided ablation is an emerging minimally invasive method to treat pancreatic cancer and premalignant pancreatic cysts. Different ablative modalities have been used including alcohol, chemotherapy agents, and radiofrequency ablation. Cumulative data over the past two decades have shown that endoscopic ablation of mucinous pancreatic cysts can lead to cyst resolution in a significant proportion of the treated cysts. Furthermore, novel data are emerging about the ability to endoscopically ablate early and locally advanced pancreatic cancer. In this review, we aim to summarize the available data on the efficacy and safety of the different EUS-ablation modalities for the management of premalignant pancreatic cysts and pancreatic cancer.

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.

Oncogenic KRAS-dependent stromal interleukin-33 directs the pancreatic microenvironment to promote tumor growth.

Pancreatic cancer is characterized by an extensive fibroinflammatory microenvironment. During carcinogenesis, normal stromal cells are converted to cytokine-high cancer associated fibroblasts (CAFs). The mechanisms underlying this conversion, including regulation and function of fibroblast-derived cytokines, are poorly understood. Thus, efforts to target CAFs therapeutically have so far failed. Here, we show that signals from epithelial cells expressing oncogenic KRAS -a hallmark pancreatic cancer mutation- activate fibroblast autocrine signaling, which drives expression of the cytokine interleukin-33 (IL-33). Stromal IL-33 expression remains high and dependent on epithelial KRAS throughout carcinogenesis; in turn, environmental stress induces IL-33 secretion. Using compartment-specific IL-33 knockout mice, we observed that lack of stromal IL-33 leads to profound reprogramming of multiple components of the pancreatic tumor microenvironment, including CAFs, myeloid cells and lymphocytes. Notably, loss of stromal IL-33 leads to an increase in CD8+ T cell infiltration and activation, and, ultimately, reduced tumor growth.

WNT signaling in the tumor microenvironment promotes immunosuppression in murine pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is associated with activation of WNT signaling. Whether this signaling pathway regulates the tumor microenvironment has remained unexplored. Through single-cell RNA sequencing of human pancreatic cancer, we discovered that tumor-infiltrating CD4+ T cells express TCF7, encoding for the transcription factor TCF1. We conditionally inactivated Tcf7 in CD4 expressing T cells in a mouse model of pancreatic cancer and observed changes in the tumor immune microenvironment, including more CD8+ T cells and fewer regulatory T cells, but also compensatory upregulation of PD-L1. We then used a clinically available inhibitor of Porcupine, a key component of WNT signaling, and observed similar reprogramming of the immune response. WNT signaling inhibition has limited therapeutic window due to toxicity, and PD-L1 blockade has been ineffective in PDA. Here, we show that combination targeting reduces pancreatic cancer growth in an experimental model and might benefit the treatment of pancreatic cancer.

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.

Analysis of donor pancreata defines the transcriptomic signature and microenvironment of early pre-neoplastic pancreatic lesions.

The adult healthy human pancreas has been poorly studied given lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathological analysis of the samples revealed PanIN lesions in most individuals irrespective of age. Using a combination of multiplex immunohistochemistry, single cell RNA sequencing, and spatial transcriptomics, we provide the first ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts, and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis. Statement of significance: The causes underlying the onset of pancreatic cancer remain largely unknown, hampering early detection and prevention strategies. Here, we show that PanIN are abundant in healthy individuals and present at a much higher rate than the incidence of pancreatic cancer, setting the stage for efforts to elucidate the microenvironmental and cell intrinsic factors that restrain, or, conversely, promote, malignant progression.

Analysis of Donor Pancreata Defines the Transcriptomic Signature and Microenvironment of Early Neoplastic Lesions.

The adult healthy human pancreas has been poorly studied given the lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors, thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathologic analysis of the samples revealed pancreatic intraepithelial neoplasia (PanIN) lesions in most individuals irrespective of age. Using a combination of multiplex IHC, single-cell RNA sequencing, and spatial transcriptomics, we provide the first-ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis. SIGNIFICANCE: Precursor lesions to pancreatic cancer are poorly characterized. We analyzed donor pancreata and discovered that precursor lesions are detected at a much higher rate than the incidence of pancreatic cancer, setting the stage for efforts to elucidate the microenvironmental and cell-intrinsic factors that restrain or, conversely, promote malignant progression. See related commentary by Hoffman and Dougan, p. 1288. This article is highlighted in the In This Issue feature, p. 1275.

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.

Using Single Cell Transcriptomics to Elucidate the Myeloid Compartment in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a 5-year survival rate of 10%. A hallmark feature of this disease is its abundant microenvironment which creates a highly immunosuppressive milieu. This is, in large part, mediated by an abundant infiltration of myeloid cells in the PDAC tumor microenvironment. Consequently, therapies that modulate myeloid function may augment the efficacy of standard of care for PDAC. Unfortunately, there is limited understanding about the various subsets of myeloid cells in PDAC, particularly in human studies. This review highlights the application of single-cell RNA sequencing to define the myeloid compartment in human PDAC and elucidate the crosstalk between myeloid cells and the other components of the tumor immune microenvironment.

Hypoxia promotes an inflammatory phenotype of fibroblasts in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive fibroinflammatory stroma and often experiences conditions of insufficient oxygen availability or hypoxia. Cancer-associated fibroblasts (CAF) are a predominant and heterogeneous population of stromal cells within the pancreatic tumor microenvironment. Here, we uncover a previously unrecognized role for hypoxia in driving an inflammatory phenotype in PDAC CAFs. We identify hypoxia as a strong inducer of tumor IL1ɑ expression, which is required for inflammatory CAF (iCAF) formation. Notably, iCAFs preferentially reside in hypoxic regions of PDAC. Our data implicate hypoxia as a critical regulator of CAF heterogeneity in PDAC.

Pancreas-specific ablation of beta1 integrin induces tissue degeneration by disrupting acinar cell polarity.

BACKGROUND & AIMS: Integrin contact with basement membrane is a major determinant of epithelial cell polarity. beta1 integrin heterodimers are the primary receptors for basement membrane in pancreatic acinar cells, which function to synthesize and directionally secrete digestive enzymes into a central lumen. Aberrant acinar secretion and exposure of the parenchyma to digestive enzyme activity lead to organ damage and pancreatitis. METHODS: beta1 integrin conditional knockout mice were crossed to Ptf1a-Cre mice to ablate beta1 integrin in the pancreas. Histopathology of aged and cerulein-treated mice were assessed by histology and immunocytochemistry. Directional secretion was determined in vitro by FM1-43 loading with cerulein stimulation. RESULTS: Pancreas-specific ablation of beta1 integrin led to progressive organ degeneration, associated with focal acinar cell necrosis and ductal metaplasia along with widespread inflammation and collagen deposition. beta1 Integrin-null pancreata were highly susceptible to cerulein-induced acute pancreatitis, displaying an enhanced level of damage with no loss in regeneration. Degenerating beta1 integrin-null pancreata were marked by disruption of acinar cell polarity. Protein kinase C epsilon, normally localized apically, was found in the cytoplasm where it can lead to intracellular digestive enzyme activation. beta1 Integrin-null acinar cells displayed indiscriminate secretion to all membrane surfaces, consistent with an observed loss of basolateral membrane localization of Munc18c, which normally prevents basal secretion of digestive enzymes. CONCLUSIONS: Ablation of beta1 integrin induces organ atrophy by disrupting acinar cell polarity and exposing the pancreatic parenchyma to digestive enzymes.

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.

Pancreatic cancer is marked by complement-high blood monocytes and tumor-associated macrophages.

Pancreatic ductal adenocarcinoma (PDA) is accompanied by reprogramming of the local microenvironment, but changes at distal sites are poorly understood. We implanted biomaterial scaffolds, which act as an artificial premetastatic niche, into immunocompetent tumor-bearing and control mice, and identified a unique tumor-specific gene expression signature that includes high expression of C1qa, C1qb, Trem2, and Chil3 Single-cell RNA sequencing mapped these genes to two distinct macrophage populations in the scaffolds, one marked by elevated C1qa, C1qb, and Trem2, the other with high Chil3, Ly6c2 and Plac8 In mice, expression of these genes in the corresponding populations was elevated in tumor-associated macrophages compared with macrophages in the normal pancreas. We then analyzed single-cell RNA sequencing from patient samples, and determined expression of C1QA, C1QB, and TREM2 is elevated in human macrophages in primary tumors and liver metastases. Single-cell sequencing analysis of patient blood revealed a substantial enrichment of the same gene signature in monocytes. Taken together, our study identifies two distinct tumor-associated macrophage and monocyte populations that reflects systemic immune changes in pancreatic ductal adenocarcinoma patients.

Inhibition of Hedgehog Signaling Alters Fibroblast Composition in Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of Hedgehog signaling in PDAC have been contradictory, with Hedgehog signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how Hedgehog pathway inhibition reprograms the PDAC microenvironment. EXPERIMENTAL DESIGN: We used a combination of pharmacologic inhibition, gain- and loss-of-function genetic experiments, cytometry by time-of-flight, and single-cell RNA sequencing to study the roles of Hedgehog signaling in PDAC. RESULTS: We found that Hedgehog signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAF) compared with inflammatory CAFs (iCAF). Sonic Hedgehog overexpression promotes tumor growth, while Hedgehog pathway inhibition with the smoothened antagonist, LDE225, impairs tumor growth. Furthermore, Hedgehog pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in regulatory T cells, consistent with increased immunosuppression. CONCLUSIONS: Hedgehog pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment.

GOT1 inhibition promotes pancreatic cancer cell death by ferroptosis.

Cancer metabolism is rewired to support cell survival in response to intrinsic and environmental stressors. Identification of strategies to target these adaptions is an area of active research. We previously described a cytosolic aspartate aminotransaminase (GOT1)-driven pathway in pancreatic cancer used to maintain redox balance. Here, we sought to identify metabolic dependencies following GOT1 inhibition to exploit this feature of pancreatic cancer and to provide additional insight into regulation of redox metabolism. Using pharmacological methods, we identify cysteine, glutathione, and lipid antioxidant function as metabolic vulnerabilities following GOT1 withdrawal. We demonstrate that targeting any of these pathways triggers ferroptosis, an oxidative, iron-dependent form of cell death, in GOT1 knockdown cells. Mechanistically, we reveal that GOT1 inhibition represses mitochondrial metabolism and promotes a catabolic state. Consequently, we find that this enhances labile iron availability through autophagy, which potentiates the activity of ferroptotic stimuli. Overall, our study identifies a biochemical connection between GOT1, iron regulation, and ferroptosis.

Targeting the Microenvironment to Overcome Gemcitabine Resistance in Pancreatic Cancer.

Pancreatic cancer is characterized by an extensive and complex microenvironment, and is resistant to both chemotherapy and immune checkpoint blockade. The study by Principe and colleagues in this issue of Cancer Research proposes a combinatorial approach based on targeting the very mechanisms of resistance to gemcitabine, a commonly used chemotherapeutic agent. The authors show that gemcitabine treatment causes profound changes in the pancreatic cancer microenvironment, including elevated TGFß signaling and immune checkpoint expression, as well as increased antigen presentation in tumor cells. Accordingly, they show that the combination of chemotherapy, TGFß signaling inhibition, and immune checkpoint blockade effectively restores antitumor immunity and results in a significant survival benefit.See related article by Principe et al., p. 3101.