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.

Adar1 deletion causes degeneration of the exocrine pancreas via Mavs-dependent interferon signaling.

Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA-binding protein that deaminates adenosine (A) to inosine (I). A-to-I editing alters post-transcriptional RNA processing, making ADAR1 a crucial regulator of gene expression. Consequently, Adar1 has been implicated in organogenesis. To determine the role of Adar1 in pancreatic development and homeostasis, we conditionally deleted Adar1 from the murine pancreas (Ptf1aCre/+; Adar1Fl/Fl). The resulting mice had stunted growth, likely due to malabsorption associated with exocrine pancreatic insufficiency. Analyses of pancreata revealed ductal cell expansion, heightened interferon-stimulated gene expression and an increased influx of immune cells. Concurrent deletion of Adar1 and Mavs, a signaling protein implicated in the innate immune pathway, rescued the degenerative phenotype and resulted in normal pancreatic development. Taken together, our work suggests that the primary function of Adar1 in the pancreas is to prevent aberrant activation of the Mavs-mediated innate immune pathway, thereby maintaining pancreatic homeostasis.

Tumor-resident microbes: the new kids on the microenvironment block.

Tumor-resident microbes (TRM) are an integral component of the tumor microenvironment (TME). TRM can influence tumor growth, distant dissemination, and response to therapies by interfering with molecular pathways in tumor cells as well as with other components of the TME. Novel technologies are improving the identification and visualization of cell type-specific microbes in the TME. The mechanisms that mediate the role of TRM at the primary tumors and metastatic sites are being elucidated. This knowledge is providing novel perspectives for targeting microbes or using microbial interventions for cancer interception or therapy.

Assessing Engraftment Following Fecal Microbiota Transplant.

Fecal Microbiota Transplant (FMT) is an FDA approved treatment for recurrent Clostridium difficile infections, and is being explored for other clinical applications, from alleviating digestive and neurological disorders, to priming the microbiome for cancer treatment, and restoring microbiomes impacted by cancer treatment. Quantifying the extent of engraftment following an FMT is important in determining if a recipient didn't respond because the engrafted microbiome didn't produce the desired outcomes (a successful FMT, but negative treatment outcome), or the microbiome didn't engraft (an unsuccessful FMT and negative treatment outcome). The lack of a consistent methodology for quantifying FMT engraftment extent hinders the assessment of FMT success and its relation to clinical outcomes, and presents challenges for comparing FMT results and protocols across studies. Here we review 46 studies of FMT in humans and model organisms and group their approaches for assessing the extent to which an FMT engrafts into three criteria: 1) Chimeric Asymmetric Community Coalescence investigates microbiome shifts following FMT engraftment using methods such as alpha diversity comparisons, beta diversity comparisons, and microbiome source tracking. 2) Donated Microbiome Indicator Features tracks donated microbiome features (e.g., amplicon sequence variants or species of interest) as a signal of engraftment with methods such as differential abundance testing based on the current sample collection, or tracking changes in feature abundances that have been previously identified (e.g., from FMT or disease-relevant literature). 3) Temporal Stability examines how resistant post-FMT recipient's microbiomes are to reverting back to their baseline microbiome. Individually, these criteria each highlight a critical aspect of microbiome engraftment; investigated together, however, they provide a clearer assessment of microbiome engraftment. We discuss the pros and cons of each of these criteria, providing illustrative examples of their application. We also introduce key terminology and recommendations on how FMT studies can be analyzed for rigorous engraftment extent assessment.

Gut epithelial Interleukin-17 receptor A signaling can modulate distant tumors growth through microbial regulation.

Microbes influence cancer initiation, progression and therapy responsiveness. IL-17 signaling contributes to gut barrier immunity by regulating microbes but also drives tumor growth. A knowledge gap remains regarding the influence of enteric IL-17-IL-17RA signaling and their microbial regulation on the behavior of distant tumors. We demonstrate that gut dysbiosis induced by systemic or gut epithelial deletion of IL-17RA induces growth of pancreatic and brain tumors due to excessive development of Th17, primary source of IL-17 in human and mouse pancreatic ductal adenocarcinoma, as well as B cells that circulate to distant tumors. Microbial dependent IL-17 signaling increases DUOX2 signaling in tumor cells. Inefficacy of pharmacological inhibition of IL-17RA is overcome with targeted microbial ablation that blocks the compensatory loop. These findings demonstrate the complexities of IL-17-IL-17RA signaling in different compartments and the relevance for accounting for its homeostatic host defense function during cancer therapy.

CD73-Dependent Adenosine Signaling through Adora2b Drives Immunosuppression in Ductal Pancreatic Cancer.

The microenvironment that surrounds pancreatic ductal adenocarcinoma (PDAC) is profoundly desmoplastic and immunosuppressive. Understanding triggers of immunosuppression during the process of pancreatic tumorigenesis would aid in establishing targets for effective prevention and therapy. Here, we interrogated differential molecular mechanisms dependent on cell of origin and subtype that promote immunosuppression during PDAC initiation and in established tumors. Transcriptomic analysis of cell-of-origin-dependent epithelial gene signatures revealed that Nt5e/CD73, a cell-surface enzyme required for extracellular adenosine generation, is one of the top 10% of genes overexpressed in murine tumors arising from the ductal pancreatic epithelium as opposed to those rising from acinar cells. These findings were confirmed by IHC and high-performance liquid chromatography. Analysis in human PDAC subtypes indicated that high Nt5e in murine ductal PDAC models overlaps with high NT5E in human PDAC squamous and basal subtypes, considered to have the highest immunosuppression and worst prognosis. Multiplex immunofluorescent analysis showed that activated CD8+ T cells in the PDAC tumor microenvironment express high levels of CD73, indicating an opportunity for immunotherapeutic targeting. Delivery of CD73 small-molecule inhibitors through various delivery routes reduced tumor development and growth in genetically engineered and syngeneic mouse models. In addition, the adenosine receptor Adora2b was a determinant of adenosine-mediated immunosuppression in PDAC. These findings highlight a molecular trigger of the immunosuppressive PDAC microenvironment elevated in the ductal cell of origin, linking biology with subtype classification, critical components for PDAC immunoprevention and personalized approaches for immunotherapeutic intervention. SIGNIFICANCE: Ductal-derived pancreatic tumors have elevated epithelial and CD8+GZM+ T-cell CD73 expression that confers sensitivity to small-molecule inhibition of CD73 or Adora2b to promote CD8+ T-cell-mediated tumor regression. See related commentary by DelGiorno, p. 977.

Cytometry of Mass for Murine Immunoprofiling.

Mass cytometry or cytometry by time-of-flight (CyTOF) is a multi-parametric analytical tool that is commonly used for simultaneous detection of more than 50 markers present either on the surface or inside the cytoplasm or nucleus of the cell. It utilizes metal-tagged antibodies and offers numerous advantages over traditional immunophenotyping techniques like flow cytometry, such as minimal overlap between channels and near zero background cellular signal. CyTOF is widely used for global immunoprofiling aimed at identification of biomarkers during cancer prevention clinical and preclinical studies that can further aid in the development of early detection markers and preventive strategies. In this unit, we describe the staining protocols and analytical tools for performing suspension CyTOF.

Mouse Models to Study Secondary Cancer Prevention.

Secondary prevention is a set of procedures involved in discovering early recurrence, local or systemic metastasis before the clinical signs or symptoms. We describe a mouse model with orthotopic pancreatic tumor implantation followed by distal pancreatectomy. The bioluminescence imaging and MRI could be used for screening the resected primary tumor recurrence and secondary cancer development. Different types of surgical procedures, chemotherapy, or immunotherapy can be engaged in reducing the metastasis potential of primary cancers. This model has been proved to be safe and easy to establish, which can mimic the clinical scenario and expand perspectives for studying the effects of tumor resection and adjuvant or neoadjuvant therapy on secondary cancer prevention.

Mouse T cell priming is enhanced by maturation-dependent stiffening of the dendritic cell cortex.

T cell activation by dendritic cells (DCs) involves forces exerted by the T cell actin cytoskeleton, which are opposed by the cortical cytoskeleton of the interacting antigen-presenting cell. During an immune response, DCs undergo a maturation process that optimizes their ability to efficiently prime naïve T cells. Using atomic force microscopy, we find that during maturation, DC cortical stiffness increases via a process that involves actin polymerization. Using stimulatory hydrogels and DCs expressing mutant cytoskeletal proteins, we find that increasing stiffness lowers the agonist dose needed for T cell activation. CD4+ T cells exhibit much more profound stiffness dependency than CD8+ T cells. Finally, stiffness responses are most robust when T cells are stimulated with pMHC rather than anti-CD3ε, consistent with a mechanosensing mechanism involving receptor deformation. Taken together, our data reveal that maturation-associated cytoskeletal changes alter the biophysical properties of DCs, providing mechanical cues that costimulate T cell activation.

Interleukin-17-induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with an immunosuppressive microenvironment that is resistant to most therapies. IL17 is involved in pancreatic tumorigenesis, but its role in invasive PDAC is undetermined. We hypothesized that IL17 triggers and sustains PDAC immunosuppression. We inhibited IL17/IL17RA signaling using pharmacological and genetic strategies alongside mass cytometry and multiplex immunofluorescence techniques. We uncovered that IL17 recruits neutrophils, triggers neutrophil extracellular traps (NETs), and excludes cytotoxic CD8 T cells from tumors. Additionally, IL17 blockade increases immune checkpoint blockade (PD-1, CTLA4) sensitivity. Inhibition of neutrophils or Padi4-dependent NETosis phenocopies IL17 neutralization. NMR spectroscopy revealed changes in tumor lactate as a potential early biomarker for IL17/PD-1 combination efficacy. Higher expression of IL17 and PADI4 in human PDAC corresponds with poorer prognosis, and the serum of patients with PDAC has higher potential for NETosis. Clinical studies with IL17 and checkpoint blockade represent a novel combinatorial therapy with potential efficacy for this lethal disease.

Integrins Modulate T Cell Receptor Signaling by Constraining Actin Flow at the Immunological Synapse.

Full T cell activation requires coordination of signals from multiple receptor-ligand pairs that interact in parallel at a specialized cell-cell contact site termed the immunological synapse (IS). Signaling at the IS is intimately associated with actin dynamics; T cell receptor (TCR) engagement induces centripetal flow of the T cell actin network, which in turn enhances the function of ligand-bound integrins by promoting conformational change. Here, we have investigated the effects of integrin engagement on actin flow, and on associated signaling events downstream of the TCR. We show that integrin engagement significantly decelerates centripetal flow of the actin network. In primary CD4+ T cells, engagement of either LFA-1 or VLA-4 by their respective ligands ICAM-1 and VCAM-1 slows actin flow. Slowing is greatest when T cells interact with low mobility integrin ligands, supporting a predominately drag-based mechanism. Using integrin ligands presented on patterned surfaces, we demonstrate that the effects of localized integrin engagement are distributed across the actin network, and that focal adhesion proteins, such as talin, vinculin, and paxillin, are recruited to sites of integrin engagement. Further analysis shows that talin and vinculin are interdependent upon one another for recruitment, and that ongoing actin flow is required. Suppression of vinculin or talin partially relieves integrin-dependent slowing of actin flow, indicating that these proteins serve as molecular clutches that couple engaged integrins to the dynamic actin network. Finally, we found that integrin-dependent slowing of actin flow is associated with reduction in tyrosine phosphorylation downstream of the TCR, and that this modulation of TCR signaling depends on expression of talin and vinculin. More generally, we found that integrin-dependent effects on actin retrograde flow were strongly correlated with effects on TCR signaling. Taken together, these studies support a model in which ligand-bound integrins engage the actin cytoskeletal network via talin and vinculin, and tune TCR signaling events by modulating actin dynamics at the IS.