CXCR4 inhibition in human pancreatic and colorectal cancers induces an integrated immune response.

Inhibition of the chemokine receptor CXCR4 in combination with blockade of the PD-1/PD-L1 T cell checkpoint induces T cell infiltration and anticancer responses in murine and human pancreatic cancer. Here we elucidate the mechanism by which CXCR4 inhibition affects the tumor immune microenvironment. In human immune cell-based chemotaxis assays, we find that CXCL12-stimulated CXCR4 inhibits the directed migration mediated by CXCR1, CXCR3, CXCR5, CXCR6, and CCR2, respectively, chemokine receptors expressed by all of the immune cell types that participate in an integrated immune response. Inhibiting CXCR4 in an experimental cancer medicine study by 1-wk continuous infusion of the small-molecule inhibitor AMD3100 (plerixafor) induces an integrated immune response that is detected by transcriptional analysis of paired biopsies of metastases from patients with microsatellite stable colorectal and pancreatic cancer. This integrated immune response occurs in three other examples of immune-mediated damage to noninfected tissues: Rejecting renal allografts, melanomas clinically responding to anti-PD1 antibody therapy, and microsatellite instable colorectal cancers. Thus, signaling by CXCR4 causes immune suppression in human pancreatic ductal adenocarcinoma and colorectal cancer by impairing the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells.

Method To Visualize the Intratumor Distribution and Impact of Gemcitabine in Pancreatic Ductal Adenocarcinoma by Multimodal Imaging.

Gemcitabine (dFdC) is a common treatment for pancreatic cancer; however, it is thought that treatment may fail because tumor stroma prevents drug distribution to tumor cells. Gemcitabine is a pro-drug with active metabolites generated intracellularly; therefore, visualizing the distribution of parent drug as well as its metabolites is important. A multimodal imaging approach was developed using spatially coregistered mass spectrometry imaging (MSI), imaging mass cytometry (IMC), multiplex immunofluorescence microscopy (mIF), and hematoxylin and eosin (H&E) staining to assess the local distribution and metabolism of gemcitabine in tumors from a genetically engineered mouse model of pancreatic cancer (KPC) allowing for comparisons between effects in the tumor tissue and its microenvironment. Mass spectrometry imaging (MSI) enabled the visualization of the distribution of gemcitabine (100 mg/kg), its phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP, and the inactive metabolite dFdU. Distribution was compared to small-molecule ATR inhibitor AZD6738 (25 mg/kg), which was codosed. Gemcitabine metabolites showed heterogeneous distribution within the tumor, which was different from the parent compound. The highest abundance of dFdCMP, dFdCDP, and dFdCTP correlated with distribution of endogenous AMP, ADP, and ATP in viable tumor cell regions, showing that gemcitabine active metabolites are reaching the tumor cell compartment, while AZD6738 was located to nonviable tumor regions. The method revealed that the generation of active, phosphorylated dFdC metabolites as well as treatment-induced DNA damage primarily correlated with sites of high proliferation in KPC PDAC tumor tissue, rather than sites of high parent drug abundance.

RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis.

Receptor-activity-modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein-coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration. Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties.

Complete loss of ATM function augments replication catastrophe induced by ATR inhibition and gemcitabine in pancreatic cancer models.

BACKGROUND: Personalised medicine strategies may improve outcomes in pancreatic ductal adenocarcinoma (PDAC), but validation of predictive biomarkers is required. Having developed a clinical trial to assess the ATR inhibitor, AZD6738, in combination with gemcitabine (ATRi/gem), we investigated ATM loss as a predictive biomarker of response to ATRi/gem in PDAC. METHODS: Through kinase inhibition, siRNA depletion and CRISPR knockout of ATM, we assessed how ATM targeting affected the sensitivity of PDAC cells to ATRi/gem. Using flow cytometry, immunofluorescence and immunoblotting, we investigated how ATRi/gem synergise in ATM-proficient and ATM-deficient cells, before assessing the impact of ATM loss on ATRi/gem sensitivity in vivo. RESULTS: Complete loss of ATM function (through pharmacological inhibition or CRISPR knockout), but not siRNA depletion, sensitised to ATRi/gem. In ATM-deficient cells, ATRi/gem-induced replication catastrophe was augmented, while phospho-Chk2-T68 and phospho-KAP1-S824 persisted via DNA-PK activity. ATRi/gem caused growth delay in ATM-WT xenografts in NSG mice and induced regression in ATM-KO xenografts. CONCLUSIONS: ATM loss augments replication catastrophe-mediated cell death induced by ATRi/gem and may predict clinical responsiveness to this combination. ATM status should be carefully assessed in tumours from patients with PDAC, since distinction between ATM-low and ATM-null could be critical in maximising the success of clinical trials using ATM expression as a predictive biomarker.

Early relapse on adjuvant gemcitabine associated with an exceptional response to 2nd line capecitabine chemotherapy in a patient with pancreatic adenosquamous carcinoma with strong intra-tumoural expression of cytidine deaminase: a case report.

BACKGROUND: Pancreatic adenosquamous carcinoma has a poor prognosis, with limited prospective trial data to guide optimal treatment. The potential impact of drug metabolism on the treatment response of patients with pancreatic adenosquamous carcinoma is largely unknown. CASE PRESENTATION: We describe the case of a 51 year old woman with pancreatic adenosquamous carcinoma who, following surgical resection, experienced early disease relapse during adjuvant gemcitabine therapy. Paradoxically, this was followed by an exceptional response to capecitabine therapy lasting 34.6 months. Strong expression of cytidine deaminase was detected within the tumour. CONCLUSIONS: This case study demonstrates that early relapse during adjuvant chemotherapy for pancreatic adenosquamous carcinoma may be compatible with a subsequent exceptional response to second line chemotherapy, an important observation given the poor overall prognosis of patients with adenosquamous carcinoma. Cytidine deaminase is predicted to inactivate gemcitabine and, conversely, catalyze capecitabine activation. We discuss strong intra-tumoural expression of cytidine deaminase as a potential mechanism to explain this patient's disparate responses to gemcitabine and capecitabine therapy, and highlight the benefit that may be gained from considering similar determinants of response to chemotherapy in clinical practice.

A quantitative FastFUCCI assay defines cell cycle dynamics at a single-cell level.

The fluorescence ubiquitination-based cell cycle indicator (FUCCI) is a powerful tool for use in live cells but current FUCCI-based assays have limited throughput in terms of image processing and quantification. Here, we developed a lentiviral system that rapidly introduced FUCCI transgenes into cells by using an all-in-one expression cassette, FastFUCCI. The approach alleviated the need for sequential transduction and characterisation, improving labelling efficiency. We coupled the system to an automated imaging workflow capable of handling large datasets. The integrated assay enabled analyses of single-cell readouts at high spatiotemporal resolution. With the assay, we captured in detail the cell cycle alterations induced by antimitotic agents. We found that treated cells accumulated at G2 or M phase but eventually advanced through mitosis into the next interphase, where the majority of cell death occurred, irrespective of the preceding mitotic phenotype. Some cells appeared viable after mitotic slippage, and a fraction of them subsequently re-entered S phase. Accordingly, we found evidence that targeting the DNA replication origin activity sensitised cells to paclitaxel. In summary, we demonstrate the utility of the FastFUCCI assay for quantifying spatiotemporal dynamics and identify its potential in preclinical drug development.

Cancer Immunotherapy Trials Underutilize Immune Response Monitoring.

Immune-related radiological and biomarker monitoring in cancer immunotherapy trials permits interrogation of efficacy and reasons for therapeutic failure. We report the results from a cross-sectional analysis of response monitoring in 685 T-cell checkpoint-targeted cancer immunotherapy trials in solid malignancies, as registered on the U.S. National Institutes of Health trial registry by October 2016. Immune-related radiological response criteria were registered for only 25% of clinical trials. Only 38% of trials registered an exploratory immunological biomarker, and registration of immunological biomarkers has decreased over the last 15 years. We suggest that increasing the utilization of immune-related response monitoring across cancer immunotherapy trials will improve analysis of outcomes and facilitate translational efforts to extend the benefit of immunotherapy to a greater proportion of patients with cancer.

Modelling of the cancer cell cycle as a tool for rational drug development: A systems pharmacology approach to cyclotherapy.

The dynamic of cancer is intimately linked to a dysregulation of the cell cycle and signalling pathways. It has been argued that selectivity of treatments could exploit loss of checkpoint function in cancer cells, a concept termed "cyclotherapy". Quantitative approaches that describe these dysregulations can provide guidance in the design of novel or existing cancer therapies. We describe and illustrate this strategy via a mathematical model of the cell cycle that includes descriptions of the G1-S checkpoint and the spindle assembly checkpoint (SAC), the EGF signalling pathway and apoptosis. We incorporated sites of action of four drugs (palbociclib, gemcitabine, paclitaxel and actinomycin D) to illustrate potential applications of this approach. We show how drug effects on multiple cell populations can be simulated, facilitating simultaneous prediction of effects on normal and transformed cells. The consequences of aberrant signalling pathways or of altered expression of pro- or anti-apoptotic proteins can thus be compared. We suggest that this approach, particularly if used in conjunction with pharmacokinetic modelling, could be used to predict effects of specific oncogene expression patterns on drug response. The strategy could be used to search for synthetic lethality and optimise combination protocol designs.

Combenefit: an interactive platform for the analysis and visualization of drug combinations.

MOTIVATION: Many drug combinations are routinely assessed to identify synergistic interactions in the attempt to develop novel treatment strategies. Appropriate software is required to analyze the results of these studies. RESULTS: We present Combenefit, new free software tool that enables the visualization, analysis and quantification of drug combination effects in terms of synergy and/or antagonism. Data from combinations assays can be processed using classical Synergy models (Loewe, Bliss, HSA), as single experiments or in batch for High Throughput Screens. This user-friendly tool provides laboratory scientists with an easy and systematic way to analyze their data. The companion package provides bioinformaticians with critical implementations of routines enabling the processing of combination data. AVAILABILITY AND IMPLEMENTATION: Combenefit is provided as a Matlab package but also as standalone software for Windows (http://sourceforge.net/projects/combenefit/). CONTACT: Giovanni.DiVeroli@cruk.cam.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Natural Products Discovered in a High-Throughput Screen Identified as Inhibitors of RGS17 and as Cytostatic and Cytotoxic Agents for Lung and Prostate Cancer Cell Lines.

Regulator of G Protein Signaling (RGS) 17 is an overexpressed promoter of cancer survival in lung and prostate tumors, the knockdown of which results in decreased tumor cell proliferation in vitro. Identification of drug-like molecules inhibiting this protein could ameliorate the RGS17's pro-tumorigenic effect. Using high-throughput screening, a chemical library containing natural products was interrogated for inhibition of the RGS17-Gαo interaction. Initial hits were verified in control and counter screens. Leads were characterized via biochemical, mass spectrometric, Western blot, microscopic, and cytotoxicity measures. Four known compounds (1-4) were identified with IC50 values ranging from high nanomolar to low micromolar. Three compounds were extensively characterized biologically, demonstrating cellular activity determined by confocal microscopy, and two compounds were assessed via ITC exhibiting high nanomolar to low micromolar dissociation constants. The compounds were found to have a cysteine-dependent mechanism of binding, verified through site-directed mutagenesis and cysteine reactivity assessment. Two compounds, sanguinarine (1) and celastrol (2), were found to be cytostatic against lung and prostate cancer cell lines and cytotoxic against prostate cancer cell lines in vitro, although the dependence of RGS17 on these phenomena remains elusive, a result that is perhaps not surprising given the multimodal cytostatic and cytotoxic activities of many natural products.

Understanding the Complexity of Porous Graphitic Carbon (PGC) Chromatography: Modulation of Mobile-Stationary Phase Interactions Overcomes Loss of Retention and Reduces Variability.

Porous graphitic carbon (PGC) is an important tool in a chromatographer's armory that retains polar compounds with mass spectrometry (MS)-compatible solvents. However, its applicability is severely limited by an unpredictable loss of retention, which can be attributed to contamination. The solutions offered fail to restore the original retention and our observations of retention time shifts of gemcitabine/metabolites on PGC are not consistent with contamination. The mobile phase affects the ionization state of analytes and the polarizable PGC surface that influences the strength of dispersive forces governing retention on the stationary phase. We hypothesized that failure to maintain the same PGC surface before and after running a gradient is a cause of the observed retention loss/variability on PGC. Herein, we optimize the choice of mobile phase solvent in a gradient program with three parts: a preparatory phase, which allows binding of analytes to column; an elution phase, which gives the required separation/peak shape; and a maintenance phase, to preserve the required retention capacity. Via liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of gemcitabine and its metabolites extracted from tumor tissue, we demonstrate reproducible chromatography on three PGC columns of different ages. This approach simplifies use of the PGC to the same level as that of a C-18 column, removes the need for column regeneration, and minimizes run times, thus allowing PGC columns to be used to their full potential.

CTGF antagonism with mAb FG-3019 enhances chemotherapy response without increasing drug delivery in murine ductal pancreas cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant desmoplasia and poor tissue perfusion. These features are proposed to limit the access of therapies to neoplastic cells and blunt treatment efficacy. Indeed, several agents that target the PDA tumor microenvironment promote concomitant chemotherapy delivery and increased antineoplastic response in murine models of PDA. Prior studies could not determine whether chemotherapy delivery or microenvironment modulation per se were the dominant features in treatment response, and such information could guide the optimal translation of these preclinical findings to patients. To distinguish between these possibilities, we used a chemical inhibitor of cytidine deaminase to stabilize and thereby artificially elevate gemcitabine levels in murine PDA tumors without disrupting the tumor microenvironment. Additionally, we used the FG-3019 monoclonal antibody (mAb) that is directed against the pleiotropic matricellular signaling protein connective tissue growth factor (CTGF/CCN2). Inhibition of cytidine deaminase raised the levels of activated gemcitabine within PDA tumors without stimulating neoplastic cell killing or decreasing the growth of tumors, whereas FG-3019 increased PDA cell killing and led to a dramatic tumor response without altering gemcitabine delivery. The response to FG-3019 correlated with the decreased expression of a previously described promoter of PDA chemotherapy resistance, the X-linked inhibitor of apoptosis protein. Therefore, alterations in survival cues following targeting of tumor microenvironmental factors may play an important role in treatment responses in animal models, and by extension in PDA patients.

Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer.

An autochthonous model of pancreatic ductal adenocarcinoma (PDA) permitted the analysis of why immunotherapy is ineffective in this human disease. Despite finding that PDA-bearing mice had cancer cell-specific CD8(+) T cells, the mice, like human patients with PDA, did not respond to two immunological checkpoint antagonists that promote the function of T cells: anti-cytotoxic T-lymphocyte-associated protein 4 (α-CTLA-4) and α-programmed cell death 1 ligand 1 (α-PD-L1). Immune control of PDA growth was achieved, however, by depleting carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). The depletion of the FAP(+) stromal cell also uncovered the antitumor effects of α-CTLA-4 and α-PD-L1, indicating that its immune suppressive activity accounts for the failure of these T-cell checkpoint antagonists. Three findings suggested that chemokine (C-X-C motif) ligand 12 (CXCL12) explained the overriding immunosuppression by the FAP(+) cell: T cells were absent from regions of the tumor containing cancer cells, cancer cells were coated with the chemokine, CXCL12, and the FAP(+) CAF was the principal source of CXCL12 in the tumor. Administering AMD3100, a CXCL12 receptor chemokine (C-X-C motif) receptor 4 inhibitor, induced rapid T-cell accumulation among cancer cells and acted synergistically with α-PD-L1 to greatly diminish cancer cells, which were identified by their loss of heterozygosity of Trp53 gene. The residual tumor was composed only of premalignant epithelial cells and inflammatory cells. Thus, a single protein, CXCL12, from a single stromal cell type, the FAP(+) CAF, may direct tumor immune evasion in a model of human PDA.

SPARC independent drug delivery and antitumour effects of nab-paclitaxel in genetically engineered mice.

DESIGN: Pharmacokinetic and pharmacodynamic parameters of cremophor-paclitaxel, nab-paclitaxel (human-albumin-bound paclitaxel, Abraxane) and a novel mouse-albumin-bound paclitaxel (m-nab-paclitaxel) were evaluated in genetically engineered mouse models (GEMMs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS), histological and biochemical analysis. Preclinical evaluation of m-nab-paclitaxel included assessment by three-dimensional high-resolution ultrasound and molecular analysis in a novel secreted protein acidic and rich in cysteine (SPARC)-deficient GEMM of pancreatic ductal adenocarcinoma (PDA). RESULTS: nab-Paclitaxel exerted its antitumoural effects in a dose-dependent manner and was associated with less toxicity compared with cremophor-paclitaxel. SPARC nullizygosity in a GEMM of PDA, Kras(G12D);p53(flox/-);p48Cre (KPfC), resulted in desmoplastic ductal pancreas tumours with impaired collagen maturation. Paclitaxel concentrations were significantly decreased in SPARC null plasma samples and tissues when administered as low-dose m-nab-paclitaxel. At the maximally tolerated dose, SPARC deficiency did not affect the intratumoural paclitaxel concentration, stromal deposition and the immediate therapeutic response. CONCLUSIONS: nab-Paclitaxel accumulates and acts in a dose-dependent manner. The interaction of plasma SPARC and albumin-bound drugs is observed at low doses of nab-paclitaxel but is saturated at therapeutic doses in murine tumours. Thus, this study provides important information for future preclinical and clinical trials in PDA using nab-paclitaxel in combination with novel experimental and targeted agents.

Effects of high-fat and high-carbohydrate diets on fat and carbohydrate oxidation and plasma metabolites in healthy cats.

High-fat (HF) or high-carbohydrate (HC) diets (30% fat, 18.9% carbohydrate; HF and 10% fat, 46.3% carbohydrate; HC) and lengths of adaptation were investigated in cats (Felis catus; 10 ± 2 months, 3.6 ± 0.3 kg). Cats randomly received each treatment for 14 days in a crossover design with a 14-day washout period between each diet. Three 22-h indirect calorimetry studies were conducted after acute (day 0), semichronic (day 4) and chronic (day 13) dietary exposure. Blood samples were collected after a 24-h fast on days 1, 5 and 14. When cats consumed the HC and HF diet, oxidation of the restricted nutrient exceeded intake while oxidation of the nutrient in excess matched intake. Mean max energy expenditure (EE) of cats consuming the HF and HC diet were 107 and 102 kcal/kg(0.67)/day and occurred at a mean of 4 and 12 h post-feeding respectively. Maximal fat (0.90 g/h) and carbohydrate (carbohydrate; 1.42 g/h) oxidation were attained at 26 min and 10.4 h post-feeding respectively. The changes observed in macronutrient oxidation and EE suggest that cats adapt whole-body nutrient metabolism in response to changes in dietary macronutrient content, but may require longer than 14 day to adapt to a macronutrient that is present at a lower concentration in the diet.

Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDA) is characterised by stromal desmoplasia and vascular dysfunction, which critically impair drug delivery. This study examines the role of an abundant extracellular matrix component, the megadalton glycosaminoglycan hyaluronan (HA), as a novel therapeutic target in PDA. METHODS: Using a genetically engineered mouse model of PDA, the authors enzymatically depleted HA by a clinically formulated PEGylated human recombinant PH20 hyaluronidase (PEGPH20) and examined tumour perfusion, vascular permeability and drug delivery. The preclinical utility of PEGPH20 in combination with gemcitabine was assessed by short-term and survival studies. RESULTS: PEGPH20 rapidly and sustainably depleted HA, inducing the re-expansion of PDA blood vessels and increasing the intratumoral delivery of two chemotherapeutic agents, doxorubicin and gemcitabine. Moreover, PEGPH20 triggered fenestrations and interendothelial junctional gaps in PDA tumour endothelia and promoted a tumour-specific increase in macromolecular permeability. Finally, combination therapy with PEGPH20 and gemcitabine led to inhibition of PDA tumour growth and prolonged survival over gemcitabine monotherapy, suggesting immediate clinical utility. CONCLUSIONS: The authors demonstrate that HA impedes the intratumoral vasculature in PDA and propose that its enzymatic depletion be explored as a means to improve drug delivery and response in patients with pancreatic cancer.

Intratumoral antigen signaling traps CD8+ T cells to confine exhaustion to the tumor site.

Immunotherapy advances have been hindered by difficulties in tracking the behaviors of lymphocytes after antigen signaling. Here, we assessed the behavior of T cells active within tumors through the development of the antigen receptor signaling reporter (AgRSR) mouse, fate-mapping lymphocytes responding to antigens at specific times and locations. Contrary to reports describing the ready egress of T cells out of the tumor, we find that intratumoral antigen signaling traps CD8+ T cells in the tumor. These clonal populations expand and become increasingly exhausted over time. By contrast, antigen-signaled regulatory T cell (Treg) clonal populations readily recirculate out of the tumor. Consequently, intratumoral antigen signaling acts as a gatekeeper to compartmentalize CD8+ T cell responses, even within the same clonotype, thus enabling exhausted T cells to remain confined to a specific tumor tissue site.

Microstructure, rheological and water mobility behaviour of plant-based protein isolates (pea and quinoa) and locust bean gum mixtures.

This work reports the impact of locust bean gum (LBG) in the continuous phase of plant-based proteins, i.e. quinoa protein (QPI) and pea protein isolates (PPI). Experimental measurements such as confocal microscopy, rheological analysis and water mobility via nuclear magnetic resonance (nmr) spin-spin relaxation time (T2) were carried out. The influence of LBG on the rheological properties of QPI and PPI is consistent with an exchange-based nmr interpretation of T2 for biopolymer and water. Addition of LBG increased the viscoelastic properties (storage and loss modulus) and shear viscosities of the mixtures. LBG interacted with both plant proteins, resulting in the formation of more dense protein networks and protein coacervates. A stronger interaction between the PPI and LBG was observed, resulting in higher shear viscosities with lower water mobility as compared to QPI:LBG formulations. Results indicated that the interaction between the protein and polysaccharide played a significant role in the microstructure, its rheological properties and consequently water mobility.

Defining the spatial distribution of extracellular adenosine revealed a myeloid-dependent immunosuppressive microenvironment in pancreatic ductal adenocarcinoma.

BACKGROUND: The prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) remains extremely poor. It has been suggested that the adenosine pathway contributes to the ability of PDAC to evade the immune system and hence, its resistance to immuno-oncology therapies (IOT), by generating extracellular adenosine (eAdo). METHODS: Using genetically engineered allograft models of PDAC in syngeneic mice with defined and different immune infiltration and response to IOT and autochthonous tumors in KPC mice we investigated the impact of the adenosine pathway on the PDAC tumor microenvironment (TME). Flow cytometry and imaging mass cytometry (IMC) were used to characterize the subpopulation frequency and spatial distribution of tumor-infiltrating immune cells. Mass spectrometry imaging (MSI) was used to visualize adenosine compartmentalization in the PDAC tumors. RNA sequencing was used to evaluate the influence of the adenosine pathway on the shaping of the immune milieu and correlate our findings to published data sets in human PDAC. RESULTS: We demonstrated high expression of adenosine pathway components in tumor-infiltrating immune cells (particularly myeloid populations) in the murine models. MSI demonstrated that extracellular adenosine distribution is heterogeneous in tumors, with high concentrations in peri-necrotic, hypoxic regions, associated with rich myeloid infiltration, demonstrated using IMC. Protumorigenic M2 macrophages express high levels of the Adora2a receptor; particularly in the IOT resistant model. Blocking the in vivo formation and function of eAdo (Adoi), using a combination of anti-CD73 antibody and an Adora2a inhibitor slowed tumor growth and reduced metastatic burden. Additionally, blocking the adenosine pathway improved the efficacy of combinations of cytotoxic agents or immunotherapy. Adoi remodeled the TME, by reducing the infiltration of M2 macrophages and regulatory T cells. RNA sequencing analysis showed that genes related to immune modulation, hypoxia and tumor stroma were downregulated following Adoi and a specific adenosine signature derived from this is associated with a poorer prognosis in patients with PDAC. CONCLUSIONS: The formation of eAdo promotes the development of the immunosuppressive TME in PDAC, contributing to its resistance to conventional and novel therapies. Therefore, inhibition of the adenosine pathway may represent a strategy to modulate the PDAC immune milieu and improve therapy response in patients with PDAC.

Direct histological processing of EUS biopsies enables rapid molecular biomarker analysis for interventional pancreatic cancer trials.

OBJECTIVE: Current practice to diagnose pancreatic cancer is accomplished by endoscopic ultrasound guided fine needle aspiration (EUS-FNA) using a cytological approach. This method is time consuming and often fails to provide suitable specimens for modern molecular analyses. Here, we compare the cytological approach with direct formalin fixation of pancreatic EUS-FNA micro-cores and evaluate the potential to perform molecular biomarker analysis on these specimen. METHODS: 130 specimens obtained by EUS-FNA with a 22G needle were processed by the standard cytological approach and compared to a separate cohort of 130 specimens that were immediately formalin fixed to preserve micro-cores of tissue prior to routine histological processing. RESULTS: We found that direct formalin fixation significantly shortened the time required for diagnosis from 3.6 days to 2.9 days (p<0.05) by reducing the average time (140 vs 33 min/case) and number of slides (9.65 vs 4.67 slides/case) for histopathological processing. Specificity and sensitivity yielded comparable results between the two approaches (82.3% vs 77% and 90.9% vs 100%). Importantly, EUS-FNA histology preserved the tumour tissue architecture with neoplastic glands embedded in stroma in 67.89% of diagnostic cases compared to 27.55% with the standard cytological approach (p < 0.001). Furthermore, micro-core samples were suitable for molecular studies including the immunohistochemical detection of intranuclear Hes1 in malignant cells, and the laser-capture microdissection-mediated measurement of Gli-1 mRNA in tumour stromal myofibroblasts. CONCLUSIONS: Direct formalin fixation of pancreatic EUS-FNA micro-cores demonstrates superiority regarding diagnostic delay, costs, and specimen suitability for molecular studies. We advocate this approach for future investigational trials in pancreatic cancer patients.