Pancreatic cancer: Advances and challenges.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers. Significant efforts have largely defined major genetic factors driving PDAC pathogenesis and progression. Pancreatic tumors are characterized by a complex microenvironment that orchestrates metabolic alterations and supports a milieu of interactions among various cell types within this niche. In this review, we highlight the foundational studies that have driven our understanding of these processes. We further discuss the recent technological advances that continue to expand our understanding of PDAC complexity. We posit that the clinical translation of these research endeavors will enhance the currently dismal survival rate of this recalcitrant disease.

Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins.

T cell infiltration into tumors is a favorable prognostic feature, but most solid tumors lack productive T cell responses. Mechanisms that coordinate T cell exclusion are incompletely understood. Here we identify hepatocyte activation via interleukin-6/STAT3 and secretion of serum amyloid A (SAA) proteins 1 and 2 as important regulators of T cell surveillance of extrahepatic tumors. Loss of STAT3 in hepatocytes or SAA remodeled the tumor microenvironment with infiltration by CD8+ T cells, while interleukin-6 overexpression in hepatocytes and SAA signaling via Toll-like receptor 2 reduced the number of intratumoral dendritic cells and, in doing so, inhibited T cell tumor infiltration. Genetic ablation of SAA enhanced survival after tumor resection in a T cell-dependent manner. Likewise, in individuals with pancreatic ductal adenocarcinoma, long-term survivors after surgery demonstrated lower serum SAA levels than short-term survivors. Taken together, these data define a fundamental link between liver and tumor immunobiology wherein hepatocytes govern productive T cell surveillance in cancer.

Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer.

Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.

Spatially confined sub-tumor microenvironments in pancreatic cancer.

Intratumoral heterogeneity is a critical frontier in understanding how the tumor microenvironment (TME) propels malignant progression. Here, we deconvolute the human pancreatic TME through large-scale integration of histology-guided regional multiOMICs with clinical data and patient-derived preclinical models. We discover "subTMEs," histologically definable tissue states anchored in fibroblast plasticity, with regional relationships to tumor immunity, subtypes, differentiation, and treatment response. "Reactive" subTMEs rich in complex but functionally coordinated fibroblast communities were immune hot and inhabited by aggressive tumor cell phenotypes. The matrix-rich "deserted" subTMEs harbored fewer activated fibroblasts and tumor-suppressive features yet were markedly chemoprotective and enriched upon chemotherapy. SubTMEs originated in fibroblast differentiation trajectories, and transitory states were notable both in single-cell transcriptomics and in situ. The intratumoral co-occurrence of subTMEs produced patient-specific phenotypic and computationally predictable heterogeneity tightly linked to malignant biology. Therefore, heterogeneity within the plentiful, notorious pancreatic TME is not random but marks fundamental tissue organizational units.

Proteogenomic characterization of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.

Targeting Pin1 renders pancreatic cancer eradicable by synergizing with immunochemotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.

Endocrine-Exocrine Signaling Drives Obesity-Associated Pancreatic Ductal Adenocarcinoma.

Obesity is a major modifiable risk factor for pancreatic ductal adenocarcinoma (PDAC), yet how and when obesity contributes to PDAC progression is not well understood. Leveraging an autochthonous mouse model, we demonstrate a causal and reversible role for obesity in early PDAC progression, showing that obesity markedly enhances tumorigenesis, while genetic or dietary induction of weight loss intercepts cancer development. Molecular analyses of human and murine samples define microenvironmental consequences of obesity that foster tumorigenesis rather than new driver gene mutations, including significant pancreatic islet cell adaptation in obesity-associated tumors. Specifically, we identify aberrant beta cell expression of the peptide hormone cholecystokinin (Cck) in response to obesity and show that islet Cck promotes oncogenic Kras-driven pancreatic ductal tumorigenesis. Our studies argue that PDAC progression is driven by local obesity-associated changes in the tumor microenvironment and implicate endocrine-exocrine signaling beyond insulin in PDAC development.

Neurons Release Serine to Support mRNA Translation in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.

Senescence-Induced Vascular Remodeling Creates Therapeutic Vulnerabilities in Pancreas Cancer.

KRAS mutant pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic response that promotes hypovascularity, immunosuppression, and resistance to chemo- and immunotherapies. We show that a combination of MEK and CDK4/6 inhibitors that target KRAS-directed oncogenic signaling can suppress PDAC proliferation through induction of retinoblastoma (RB) protein-mediated senescence. In preclinical mouse models of PDAC, this senescence-inducing therapy produces a senescence-associated secretory phenotype (SASP) that includes pro-angiogenic factors that promote tumor vascularization, which in turn enhances drug delivery and efficacy of cytotoxic gemcitabine chemotherapy. In addition, SASP-mediated endothelial cell activation stimulates the accumulation of CD8+ T cells into otherwise immunologically "cold" tumors, sensitizing tumors to PD-1 checkpoint blockade. Therefore, in PDAC models, therapy-induced senescence can establish emergent susceptibilities to otherwise ineffective chemo- and immunotherapies through SASP-dependent effects on the tumor vasculature and immune system.

Tumor Microbiome Diversity and Composition Influence Pancreatic Cancer Outcomes.

Most patients diagnosed with resected pancreatic adenocarcinoma (PDAC) survive less than 5 years, but a minor subset survives longer. Here, we dissect the role of the tumor microbiota and the immune system in influencing long-term survival. Using 16S rRNA gene sequencing, we analyzed the tumor microbiome composition in PDAC patients with short-term survival (STS) and long-term survival (LTS). We found higher alpha-diversity in the tumor microbiome of LTS patients and identified an intra-tumoral microbiome signature (Pseudoxanthomonas-Streptomyces-Saccharopolyspora-Bacillus clausii) highly predictive of long-term survivorship in both discovery and validation cohorts. Through human-into-mice fecal microbiota transplantation (FMT) experiments from STS, LTS, or control donors, we were able to differentially modulate the tumor microbiome and affect tumor growth as well as tumor immune infiltration. Our study demonstrates that PDAC microbiome composition, which cross-talks to the gut microbiome, influences the host immune response and natural history of the disease.

Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer.

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

Expression of the membrane tetraspanin claudin 18 on cancer cells promotes T lymphocyte infiltration and antitumor immunity in pancreatic cancer.

Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating T cell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune-infiltrated from immune-depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased T cell activation. Our data reveal an immune role for CLDN18 in orchestrating T cell infiltration and shaping the tumor immune contexture.

A gut response: Modulating chemotherapy efficacy with microbial metabolites.

Despite clinical advances, chemotherapy largely fails in metastatic cancers. Commensal bacteria can indicate chemotherapy efficacy. In a recent issue of Nature, Tintelnot et al.1 demonstrate that bacterial metabolite 3-IAA amplifies chemotherapy outcomes via autophagy pathways in metastatic pancreatic ductal adenocarcinoma.

Enhancer Reprogramming Promotes Pancreatic Cancer Metastasis.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human malignancies, owing in part to its propensity for metastasis. Here, we used an organoid culture system to investigate how transcription and the enhancer landscape become altered during discrete stages of disease progression in a PDA mouse model. This approach revealed that the metastatic transition is accompanied by massive and recurrent alterations in enhancer activity. We implicate the pioneer factor FOXA1 as a driver of enhancer activation in this system, a mechanism that renders PDA cells more invasive and less anchorage-dependent for growth in vitro, as well as more metastatic in vivo. In this context, FOXA1-dependent enhancer reprogramming activates a transcriptional program of embryonic foregut endoderm. Collectively, our study implicates enhancer reprogramming, FOXA1 upregulation, and a retrograde developmental transition in PDA metastasis.

Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity.

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ+CD8+ T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.

γδ T Cells Support Pancreatic Oncogenesis by Restraining αß T Cell Activation.

Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated γδT cell population, which constituted ∼40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of γδT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of γδT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of αßT cells. Although αßT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon γδT cell ablation. PDA-infiltrating γδT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in γδT cells enhanced CD4(+) and CD8(+) T cell infiltration and immunogenicity and induced tumor protection suggesting that γδT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe γδT cells as central regulators of effector T cell activation in cancer via novel cross-talk.

Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation.

Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRAS(G12D)) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRAS(G12D) signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRAS(G12D) engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRAS(G12D). Consequently, reciprocal KRAS(G12D) produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRAS(G12D) alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. VIDEO ABSTRACT.

Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer.

Activating KRAS mutations (KRAS*) in pancreatic ductal adenocarcinoma (PDAC) drive anabolic metabolism and support tumor maintenance. KRAS* inhibitors show initial antitumor activity followed by recurrence due to cancer cell-intrinsic and immune-mediated paracrine mechanisms. Here, we explored the potential role of cancer-associated fibroblasts (CAFs) in enabling KRAS* bypass and identified CAF-derived NRG1 activation of cancer cell ERBB2 and ERBB3 receptor tyrosine kinases as a mechanism by which KRAS*-independent growth is supported. Genetic extinction or pharmacological inhibition of KRAS* resulted in up-regulation of ERBB2 and ERBB3 expression in human and murine models, which prompted cancer cell utilization of CAF-derived NRG1 as a survival factor. Genetic depletion or pharmacological inhibition of ERBB2/3 or NRG1 abolished KRAS* bypass and synergized with KRASG12D inhibitors in combination treatments in mouse and human PDAC models. Thus, we found that CAFs can contribute to KRAS* inhibitor therapy resistance via paracrine mechanisms, providing an actionable therapeutic strategy to improve the effectiveness of KRAS* inhibitors in PDAC patients.

It's a SMAD/SMAD World.

DPC4/SMAD4 mutations are associated with aggressive pancreatic cancer. In this issue of Cell, Whittle et al. demonstrate that Runx3 expression combined with Dpc4/Smad4 status can predict the metastatic propensity of pancreatic tumors, providing valuable guidance for personalized therapy for patients with pancreatic cancer.

Organoid models of human and mouse ductal pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.