Analysis of single nuclear chromatin accessibility reveals unique myeloid populations in human pancreatic ductal adenocarcinoma.

BACKGROUND: A better understanding of the pancreatic ductal adenocarcinoma (PDAC) immune microenvironment is critical to developing new treatments and improving outcomes. Myeloid cells are of particular importance for PDAC progression; however, the presence of heterogenous subsets with different ontogeny and impact, along with some fluidity between them, (infiltrating monocytes vs. tissue-resident macrophages; M1 vs. M2) makes characterisation of myeloid populations challenging. Recent advances in single cell sequencing technology provide tools for characterisation of immune cell infiltrates, and open chromatin provides source and function data for myeloid cells to assist in more comprehensive characterisation. Thus, we explore single nuclear assay for transposase accessible chromatin (ATAC) sequencing (snATAC-Seq), a method to analyse open gene promoters and transcription factor binding, as an important means for discerning the myeloid composition in human PDAC tumours. METHODS: Frozen pancreatic tissues (benign or PDAC) were prepared for snATAC-Seq using 10× Chromium technology. Signac was used for preliminary analysis, clustering and differentially accessible chromatin region identification. The genes annotated in promoter regions were used for Gene Ontology (GO) enrichment and cell type annotation. Gene signatures were used for survival analysis with The Cancer Genome Atlas (TCGA)-pancreatic adenocarcinoma (PAAD) dataset. RESULTS: Myeloid cell transcription factor activities were higher in tumour than benign pancreatic samples, enabling us to further stratify tumour myeloid populations. Subcluster analysis revealed eight distinct myeloid populations. GO enrichment demonstrated unique functions for myeloid populations, including interleukin-1b signalling (recruited monocytes) and intracellular protein transport (dendritic cells). The identified gene signature for dendritic cells influenced survival (hazard ratio = .63, p = .03) in the TCGA-PAAD dataset, which was unique to PDAC. CONCLUSIONS: These data suggest snATAC-Seq as a method for analysis of frozen human pancreatic tissues to distinguish myeloid populations. An improved understanding of myeloid cell heterogeneity and function is important for developing new treatment targets in PDAC.

Pancreatectomy Induces Cancer-Promoting Neutrophil Extracellular Traps.

BACKGROUND: Neutrophil extracellular traps (NETs) occur when neutrophil chromatin is decondensed and extruded into the extracellular space in a web-like structure. Originally described as an anti-microbial function, this process has been implicated in the pathogenesis of pancreatic disease. In addition, NETs are upregulated during physiologic wound-healing and coagulation. This study evaluated how the inflammatory response to pancreatic surgery influences NET formation. METHODS: For this study, 126 patients undergoing pancreatectomy gave consent before participation. Plasma was collected at several time points (preoperatively and through the postoperative outpatient visit). Plasma levels of NET markers, including cell-free DNA (cfDNA), citrullinated histone H3 (CitH3), interleukin (IL)-8, IL-6, and granulocyte colony-stimulating factor (G-CSF) were measured using enzyme-linked immunosorbent assay (ELISA). Patient clinical data were retrospectively collected from a prospectively maintained database. RESULTS: After pancreatic resection, NET markers (cfDNA and CitH3) were elevated, peaking on postoperative days 3 and 4. This increase in NETs was due to an inherent change in neutrophil biology. Postoperatively, NET-inducing cytokines (IL-8, IL-6, and G-CSF) were increased, peaking early in the postoperative course. The patients undergoing the robotic approach had a reduction in NETs during the postoperative period compared with those who underwent the open approach. The patients who experienced a pancreatic leak had an increase in NET markers during the postoperative period. CONCLUSIONS: Pancreatectomy induces cancer-promoting NET formation. The minimally invasive robotic approach may induce fewer NETs, although the current analysis was limited by selection bias. Pancreatic leak resulted in increased NETs. Further study into the potential for NET inhibition during the perioperative period is warranted.

Optimizing the synthesis of interleukin-12-loaded PLGA nanospheres (rmIL-12ns) via ultrasonication for treatment of metastatic osteosarcoma.

Clinical trials exploring bolus intravenous delivery of interleukin-12 (IL-12) for treatment of solid tumors ultimately failed due to lack of clinical response and severe dose-limiting toxicities. The present study was conducted to evaluate whether recombinant murine IL-12 (rmIL-12) could be successfully encapsulated within Poly (D, l-lactide-co-glycolide) (PLGA) nanospheres (rmIL-12ns) for safe and effective systemic delivery at pharmacologic scale. Optimal fabrication of rmIL-12ns occurs with dichloromethane as the organic solvent and emulsion formation via ultrasonication at 50% power (250 W sonicator) for 10 s (50W10s). We then determined whether utilization of synthesis modifiers including fetal bovine serum (FBS), magnesium hydroxide [Mg(OH)2 ], trehalose, or the surfactants polysorbate 80 and Span 60 alone or in combination could increase the encapsulation efficiency (EE) and/or modify the burst elution profile characteristic of the 50W10s rmIL-12ns formulation. The greatest EEs compared to the unmodified formulation were measured with modifications containing the surfactants polysorbate 80 and Span 60 (surfactant: 28.3 ± 6.10%, p = 0.29 and Surf/FBS: 85.4 ± 2.19%, p = 0.039). The Surf/FBS formulation was further modified for in vivo murine injection by substituting FBS with mouse serum albumin (MSA). The resulting Surf/MSA rmIL-12ns were then characterized before delivery at three doses (0.1, 1, and 10 mg rmIL-12ns) in our established murine model of metastatic osteosarcoma to assess efficacy. Preliminary results suggested no evidence of disease with delivery of the 0.1 mg dose in 75% of mice (3 of 4) versus a nontreated historical control (2 of 34).

Synthesis, Characterization, and In Vivo Cytokinome Profile of IL-12-Loaded PLGA Nanospheres.

We report the successful encapsulation and elution of recombinant murine IL-12 (rmIL-12) from poly(lactide-co-glycolic) acid (PLGA) nanospheres (IL-12-NS) synthesized using the double emulsion/solvent evaporation (DESE) technique with microsphere depletion through ultracentrifugation. Images obtained with scanning electron microscopy (SEM) showcased a characteristic spherical shape with a mean particle diameter of 138.1 ± 10.8 nm and zeta potential of -15.1 ± 1.249 mV. These values suggest minimal flocculation when in solution, which was reflected in an in vivo biodistribution study that reported no observed morbidity/mortality. Encapsulation efficiency (EE) was determined to be 0.101 ± 0.009% with average particle concentration obtained per batch of 1.66 × 109 ± 4.45 × 108 particles/mL. Disparate zeta (ζ) potentials obtained from both protein-loaded and protein-unloaded batches suggested surface adsorption of protein, and confocal microscopy of BSA-FITC-loaded nanospheres confirmed the presence of protein within the polymeric shell. Furthermore, elution of rmIL-12 from IL-12-NS at a concentration of 500 million particles/mL was characterized using enzyme-linked immunosorbent assay (ELISA). When IL-12-NS was administered in vivo to female BALB/c mice through retroorbital injection, IL-12-NS produced a favorable systemic cytokine profile for tumoricidal activity within the peripheral blood. Whereas IFN-γ nadir occurred at 72 hours, levels recovered quickly and displayed positive correlations postburst out to 25 days postinjection. IL-12-NS administration induced proinflammatory changes while prompting minimal counterregulatory increases in anti-inflammatory IL-10 and IL-4 cytokine levels. Further, while IL-6 levels increased to 30 folds of the baseline during the burst phase, they normalized by 72 hours and trended negatively throughout the sill phase. Similar trends were observed with IL-1ß and CXCL-1, suggesting a decreased likelihood of progression to a systemic inflammatory response syndrome-like state. As IL-12-NS delivers logarithmically lower amounts of IL-12 than previously administered during human clinical trials, our data reflect the importance of IL-12-NS which safely create a systemic immunostimulatory environment.

Macrophage and Neutrophil Interactions in the Pancreatic Tumor Microenvironment Drive the Pathogenesis of Pancreatic Cancer.

Despite modest improvements in survival in recent years, pancreatic adenocarcinoma remains a deadly disease with a 5-year survival rate of only 9%. These poor outcomes are driven by failure of early detection, treatment resistance, and propensity for early metastatic spread. Uncovering innovative therapeutic modalities to target the resistance mechanisms that make pancreatic cancer largely incurable are urgently needed. In this review, we discuss the immune composition of pancreatic tumors, including the counterintuitive fact that there is a significant inflammatory immune infiltrate in pancreatic cancer yet anti-tumor mechanisms are subverted and immune behaviors are suppressed. Here, we emphasize how immune cell interactions generate tumor progression and treatment resistance. We narrow in on tumor macrophage (TAM) spatial arrangement, polarity/function, recruitment, and origin to introduce a concept where interactions with tumor neutrophils (TAN) perpetuate the microenvironment. The sequelae of macrophage and neutrophil activities contributes to tumor remodeling, fibrosis, hypoxia, and progression. We also discuss immune mechanisms driving resistance to standard of care modalities. Finally, we describe a cadre of treatment targets, including those intended to overcome TAM and TAN recruitment and function, to circumvent barriers presented by immune infiltration in pancreatic adenocarcinoma.

Applying Osteosarcoma Immunology to Understand Disease Progression and Assess Immunotherapeutic Response.

Osteosarcoma, the most common malignant bone tumor in children and adolescents, remains a complicated disease to treat; no new treatments have been developed in more than three decades. Due to the importance of the immune system in osteosarcoma disease progression, immunotherapeutic strategies have been explored to potentially improve long-term survival. However, most immunotherapeutics have not reached the level of success hoped would occur in this disease. Understanding the immune system in osteosarcoma will be key to optimizing treatments and improving patient outcomes. Therefore, immunophenotyping can be used as a very powerful tool to help better understand the complexity of the immune response seen in osteosarcoma and in the use of immunotherapy in this malignancy. This book chapter will provide an overview of the known immune responses seen in this disease and potential developments for the future of immunophenotyping. Indeed, it appears that being able to track the immune system throughout the disease and treatment of patients with osteosarcoma could allow for a personalized approach to immunotherapy.