Search

Immune expression profile of macrophages isolated from PDAC tumors.

Immune checkpoint characterization of PDAC tumors.

Effect of PI3Kγ inhibitors on PDAC growth and survival.

Effect of PI3Kγ inhibition on immune cell populations.

Intravital imaging of orthotopic PDAC growth and spread.

Effect of macrophage secrete factors on PDAC cell migration in vitro.

Time course of PDAC tumor inflammation.

Supplementary Methods, Figure Legends

The ability of disseminated cancer cells to evade the immune response is a critical step for efficient metastatic progression. Protection against an immune attack is often provided by the tumor microenvironment that suppresses and excludes cytotoxic CD8+ T cells. Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive metastatic disease with unmet needs, yet the immunoprotective role of the metastatic tumor microenvironment in pancreatic cancer is not completely understood. In this study, we find that macrophage-derived granulin contributes to cytotoxic CD8+ T-cell exclusion in metastatic livers. Granulin expression by macrophages was induced in response to colony-stimulating factor 1. Genetic depletion of granulin reduced the formation of a fibrotic stroma, thereby allowing T-cell entry at the metastatic site. Although metastatic PDAC tumors are largely resistant to anti–PD-1 therapy, blockade of PD-1 in granulin-depleted tumors restored the antitumor immune defense and dramatically decreased metastatic tumor burden. These findings suggest that targeting granulin may serve as a potential therapeutic strategy to restore CD8+ T-cell infiltration in metastatic PDAC, thereby converting PDAC metastatic tumors, which are refractory to immune checkpoint inhibitors, into tumors that respond to immune checkpoint inhibition therapies.Significance: These findings uncover a mechanism by which metastatic PDAC tumors evade the immune response and provide the rationale for targeting granulin in combination with immune checkpoint inhibitors for the treatment of metastatic PDAC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4253/F1.large.jpg. Cancer Res; 78(15); 4253–69. ©2018 AACR.

Fig. S1. Primary M2-like macrophages express IGF-1 and IGF-2. Fig. S2. In the absence of chemotherapy, addition of MCM, IGF blockade or recombinant IGF does not affect proliferation or survival of pancreatic cancer cells. Fig.S3. MCM enhances resistance of pancreatic cancer cells to gemcitabine, 5-FU and paclitaxel in an IGF-dependent manner. Fig. S4. Biopsies from pancreatic cancer patients express activated Insulin and IGF1 receptors and are infiltrated by macrophages. Fig. S5. Mouse orthotopic pancreatic tumors are rich in macrophages and myofibroblasts. Fig. S6. Tumors from KPC mouse model express activated Insulin/IGF1 receptors. Fig. S7. Gating strategy used to FACS-sort tumor cells, immune cells and stromal cells. Fig. S8. Primary pancreatic myofibroblasts secrete IGF-1 and IGF-2. Fig. S9. IGF-1 and IGF-2 are expressed in the tumor microenvironment of PDA patients. Fig. S10. Gating strategies used to analyse tumor infiltrated immune cell populations. Fig. S11. IGF blockade decreases activation of Insulin and IGF1 receptors in pancreatic tumors. Fig. S12. Combination of 5-FU and paclitaxel with BI 836845 in a syngeneic orthotopic pancreatic cancer model.

Supplementary Figure legends. Table S1. Clinical information from 40 consented PDA patient samples. Table S2. Clinical information from 16 consented PDA patient samples. Supplementary Materials and Methods.

Files in this Data Supplement: - Supplementary Figures. Figure S1: Loss of CD8+ T cell numbers during metastatic progression in PDAC patients and genetically engineered mouse model of pancreatic cancer. Figure S2: Colony stimulating factor 1 (CSF-1) is expressed by disseminated PDAC cells, and its cognate receptor CSF-1R is expressed by metastasis associated macrophages. Figure S3: Pharmacological blockade of CSF-1R using BLZ945 reprograms MAMs, restores T cell infiltration, and reduces metastasis. Figure S4: CSF-1 inhibition reduces desmoplasia, enhances CD8+ T cell infiltration and sensitizes metastatic PDAC to αPD-1 treatment. Figure S5: Administration of Digoxin, a transcriptional inhibitor of HIF-1α, promotes M1-like MAM reprogramming and reduces Granulin level in vivo. Figure S6: αPD-1 therapy dramatically reduces metastatic tumor burden in the absence of granulin, an effect which depends on a high abundance of immune stimulatory MAMs. Figure S7: Reduction in liver fibrosis improves CD8+ T cell infiltration and enhances αPD-1 therapy in metastatic pancreatic cancer. Figure S8: Macrophage-derived granulin drives resistance to immune checkpoint inhibition in metastatic pancreatic cancer.

Tumor-associated macrophages (TAM) and myofibroblasts are key drivers in cancer that are associated with drug resistance in many cancers, including pancreatic ductal adenocarcinoma (PDAC). However, our understanding of the molecular mechanisms by which TAM and fibroblasts contribute to chemoresistance is unclear. In this study, we found that TAM and myofibroblasts directly support chemoresistance of pancreatic cancer cells by secreting insulin-like growth factors (IGF) 1 and 2, which activate insulin/IGF receptors on pancreatic cancer cells. Immunohistochemical analysis of biopsies from patients with pancreatic cancer revealed that 72% of the patients expressed activated insulin/IGF receptors on tumor cells, and this positively correlates with increased CD163+ TAM infiltration. In vivo, we found that TAM and myofibroblasts were the main sources of IGF production, and pharmacologic blockade of IGF sensitized pancreatic tumors to gemcitabine. These findings suggest that inhibition of IGF in combination with chemotherapy could benefit patients with PDAC, and that insulin/IGF1R activation may be used as a biomarker to identify patients for such therapeutic intervention. Cancer Res; 76(23); 6851–63. ©2016 AACR.

PDF file - 74K

Tumor-associated macrophages promote tumor growth by stimulating angiogenesis and suppressing antitumor immunity. Thus, therapeutics that inhibit macrophage recruitment to tumors may provide new avenues for cancer therapy. In this study, we showed how chemoattractants stromal cell-derived growth factor 1 alpha (SDF-1α) and interleukin 1 beta (IL-1β) collaborate with myeloid cell integrin-α4β1 to promote tumor inflammation and growth. We found that SDF-1α and IL-1β are highly expressed in the microenvironments of murine lung, pancreatic, and breast tumors; surprisingly, SDF-1α was expressed only by tumor cells, whereas IL-1β was produced only by tumor-derived granulocytes and macrophages. In vivo, both factors directly recruited proangiogenic macrophages to tissues, whereas antagonists of both factors suppressed tumor inflammation, angiogenesis, and growth. Signals induced by IL-1β and SDF-1α promoted the interaction of talin and paxillin with the cytoplasmic tails of integrin-α4β1, thereby stimulating myeloid cell adhesion to endothelium in vitro and in vivo. Inhibition of integrin-α4β1, SDF-1α, or IL-1β was sufficient to block tumor inflammation and growth, and the combined blockade of these molecules greatly accentuated these effects. Furthermore, antagonists of integrin-α4β1 inhibited chemotherapy-induced tumor inflammation and acted synergistically with chemotherapeutic agents to suppress tumor inflammation and growth. These results show that targeting myeloid cell recruitment mechanisms can be an effective approach to suppress tumor progression. Cancer Res; 71(22): 6965–75. ©2011 AACR.

Supplementary Figures 1-10 from Integrin α4β1 Signaling Is Required for Lymphangiogenesis and Tumor Metastasis

PDF file - 1.24MB

International audience; Sampling of information is thought to be an important aspect of explorative behaviour. Evidence for it has been gained in behavioural assessments of a variety of overt and covert cognitive domains, including sensation, attention, memory, eye movements and dexterity. A common aspect across many findings is that sampling tends to exhibit a rhythmicity at low frequencies (theta, 4–8 Hz; alpha, 9–12 Hz). Neurophysiological investigations in a wide range of species, including rodents, non-human primates and humans have demonstrated the presence of sampling related neural oscillations in a number of brain areas ranging from early sensory cortex, hippocampus to high-level cognitive areas. However, to assess whether rhythmic sampling represents a general aspect of exploratory behaviour one must critically evaluate the task parameters, and their potential link with neural oscillations. Here we focus on sampling during attentive vision to present an overview on the experimental conditions that are used to investigate rhythmic sampling and associated oscillatory brain activity in this domain. This review aims to (1) provide guidelines to efficiently quantify behavioural rhythms, (2) compare results from human and non-human primate studies and (3) argue that the underlying neural mechanisms of sampling can co-occur in both sensory and high-level areas.

Vision rests on computations that primarily rely on the parvocellular and magnocellular geniculate relay of retinal signals to V1. Secondary pathways involving superior colliculus, koniocellular lateral geniculate nucleus and pulvinar and their V1-bypassing projections to higher order cortex are known to exist. While they may form an evolutionary old visual system, their contribution to perception and visually guided behaviour remain largely obscure. Recent developments in tract tracing and circuit manipulation technologies provide new insights. Here we discuss how secondary visual pathways mediate residual vision (blindsight) after V1 injury by relaying signals directly into higher order cortical areas. We contrast these findings on blindsight with new studies on dyslexia suggesting that dysfunction of secondary visual pathways might contribute to dyslexic's perceptual difficulties. Emerging from these considerations, secondary visual pathways involving koniocellular LGN may be critical for detection of visual change, whereas pulvinar function appears more linked to visuomotor planning.

In order for organisms to survive, they need to detect rewarding stimuli, for example, food or a mate, in a complex environment with many competing stimuli. These rewarding stimuli should be detected even if they are nonsalient or irrelevant to the current goal. The value-driven theory of attentional selection proposes that this detection takes place through reward-associated stimuli automatically engaging attentional mechanisms. But how this is achieved in the brain is not very well understood. Here, we investigate the effect of differential reward on the multiunit activity in visual area V4 of monkeys performing a perceptual judgment task. Surprisingly, instead of finding reward-related increases in neural responses to the perceptual target, we observed a large suppression at the onset of the reward indicating cues. Therefore, while previous research showed that reward increases neural activity, here we report a decrease. More suppression was caused by cues associated with higher reward than with lower reward, although neither cue was informative about the perceptually correct choice. This finding of reward-associated neural suppression further highlights normalization as a general cortical mechanism and is consistent with predictions of the value-driven attention theory.

Neuronal activity in visual area V4 is well known to be modulated by selective attention, and there are reports on V4 lesions leading to attentional deficits. However, it remains unclear whether V4 microstimulation can elicit attentional benefits. To test this hypothesis, we performed local microstimulation in area V4 and explored its spatial and time dynamics in two macaque monkeys performing a visual detection task. Microstimulation was delivered via chronically implanted multi-electrode arrays. We found that microstimulation increases average performance by 35% and reduces luminance detection thresholds by −30%. This benefit critically depends on the onset of microstimulation relative to the stimulus, consistent with known dynamics of endogenous attention. These results show that local microstimulation of V4 can improve behavior and highlight the critical role of V4 for attention.

Visual perception is often not homogenous across the visual field and can vary depending on situational demands. The reasons behind this inhomogeneity are not clear. Here we show that directing attention that is consistent with a western reading habit from left to right, results in a ~32% higher sensitivity to detect transient visual events in the right hemifield. This right visual field advantage was largely reduced in individuals with reading difficulties from developmental dyslexia. Similarly, visual detection became more symmetric in skilled readers, when attention was guided opposite to the reading pattern. Taken together, these findings highlight a higher sensitivity in the right visual field for detecting the onset of sudden visual events that is well accounted for by left hemisphere dominated reading habit.

Developing optogenetics in non-human primates (NHPs) is essential for translating its successful implementation in rodents to clinical applications in humans. However, information about how optogenetics influences the primate cortex remains limited. Here, we evaluate how optogenetic stimulation of the primate primary visual cortex (V1) affects local and large-scale network activation concerned with visual perception. To this end we injected an optogenetic construct (AAV9-hSyn-ChR2-eYFP) into the V1 cortex of four macaque monkeys (macaca mulatta) and measured the effects of optogenetic V1 stimulation using functional magnetic resonance imaging (fMRI), laminar electrophysiology, and behavioural assessment. In three macaques, blood-oxygen-dependent (BOLD) fMRI activity could be reliably elicited with optogenetic stimulation in V1 and several connected extrastriate brain areas, including V2/V3, motion-sensitive area MT and the frontal-eye-fields (FEF), in particular when pulsed stimulation at 40 Hz was applied. BOLD modulation was associated with consistent neural spiking activity measured in V1 of two macaques. More detailed analysis revealed strongest neuronal activation in layer 4B and infragranular layers, which tightly reflected the histological expression pattern of the optogenetic construct in V1. Driving this visual network proved sufficient to elicit a visual percept (‘phosphene’) in one macaque during a perceptual choice task. Taken together, our findings reveal the laminar and large-cortical activation pattern related to visual phosphene generation and emphasize the need for further improving optogenetic methods in NHPs as a step towards applications in humans.

ObjectivePancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease and cytotoxic chemotherapy is the standard of care treatment for patients with advanced disease. Here, we investigate how the microenvironment in PDAC liver metastases reacts to chemotherapy and its role in metastatic disease progression post-treatment, an area which is poorly understood.DesignThe impact of chemotherapy on metastatic disease progression and immune cell infiltrates was characterised using flow and mass cytometry combined with transcriptional and histopathological analysis in experimental PDAC liver metastases mouse models. Findings were validated in patient derived liver metastases and in an autochthonous PDAC mouse model. Human and murine primary cell cocultures and ex vivo patient-derived liver explants were deployed to gain mechanistical insights on whether and how chemotherapy affects the metastatic tumour microenvironment.ResultsWe show that in vivo, chemotherapy induces an initial infiltration of proinflammatory macrophages into the liver and activates cytotoxic T cells, leading only to a temporary restraining of metastatic disease progression. However, after stopping treatment, neutrophils are recruited to the metastatic liver via CXCL1 and 2 secretion by metastatic tumour cells. These neutrophils express growth arrest specific 6 (Gas6) which leads to AXL receptor activation on tumour cells enabling their regrowth. Disruption of neutrophil infiltration or inhibition of the Gas6/AXL signalling axis in combination with chemotherapy inhibits metastatic growth. Chemotherapy increases Gas6 expression in circulating neutrophils from patients with metastatic pancreatic cancer and recombinant Gas6 is sufficient to promote tumour cell proliferation ex vivo, in patient-derived metastatic liver explants.ConclusionCombining chemotherapy with Gas6/AXL or neutrophil targeted therapy could provide a therapeutic benefit for patients with metastatic pancreatic cancer.

Natural vision involves the activation of a wide range of higher-level regions processing objects, motion, faces and actions. Here, we pursue a data-driven approach to explore how higher-level visual processes relate to the underlying structural and functional connectivity. Using a free-viewing paradigm in four awake rhesus macaque monkeys, we investigate how different visual scenes change functional connectivity. Additionally, we explore how such functional connectivity, as measured through fMRI, is related to the structural connectivity, as measured through diffusion weighted imaging. At first, we evaluate the consistency of the elicited free-viewing pattern using standard analytical techniques. We also evaluate the underlying structural connectivity via diffusion data by tracking white matter bundle projections from the visual cortex. We then reconstruct free-viewing and structural networks and quantify their properties. Centrality measures over the entire fMRI time-series revealed a consistent functional network engaged during free-viewing that included widespread hub regions across frontal (FEF, 46v), parietal (LIP, Tpt), and occipitotemporal cortex (MT, V4 and TE) among others. Interestingly, a small number of highly-weighted and long-length inter-hemispheric connections indicated the presence of long-range integrative properties during free-viewing. We hypothesized that during free-viewing, networks had the capacity to change their local and distal connections depending on the on-going changes in visual scenes. To capture these network dynamics, we depart from the static modular architecture of the structural networks and demonstrate that hubs in free-viewing networks reorganize according to the presence of objects, motion, and faces in the movie scenes indicating poly-functional properties. Lastly, we compare each NHP subject network and observe high consistency between individuals across the same network type with closer correspondence between structural networks (e.g., diffusion based and those partially assembled from tract-tracing). In summary, our network analyses revealed ongoing changes in large-scale functional organization present during free-viewing in the macaque monkey and highlight the advantages of multi-contrast imaging in awake monkeys for investigating dynamical processes in visual cognition. To further promote the use of naturalistic free-viewing paradigms and increase the development of macaque neuroimaging resources, we share our datasets in the PRIME-DE consortium.

Highlights • A non-human primate protective head cap that promotes wound healing after cranial implants. • Use of the head cap reduced wound dehiscence and the need to re-suture surgical wounds. • The head cap is easily adjustable to cover most primate cranial implants. • The head cap facilitates primate cranial implant wound management in neuroscience., Background Neuroscience studies with macaque monkeys may require cranial implants to stabilize the head or gain access to the brain for scientific purposes. Wound management that promotes healing after the cranial implant surgery in non-human primates can be difficult as it is not necessarily possible to cover the wound margins. New method Here, we developed an easily modifiable head cap that protects the sutured skin margins after cranial implant surgery and contributes to wound healing. The protective head cap was developed in response to monkeys picking at sutured skin margins around an implant, complicating healing. The user-friendly protective cap, made from Klarity- R™ Sheet (3.2 mm thick with 36 % or 42 % perforation) is affixed to the implant post-surgically. Once secured and while the monkey is still anesthetized, the plastic sheeting is molded around the implant. The protective head cap restricts the monkey’s finger access to its’ wound margins while allowing air to circulate to promote wound healing. Results and comparison with existing methods Across two UK primate facilities, the protective head cap promoted wound healing. In monkeys that did not wear the head cap, re-suturing was necessary in ∼30 % of cases. In contrast, none of the monkeys that wore the head cap required re-suturing. The monkeys wearing the head cap also had reduced numbers of days of prescribed antibiotics and analgesia. Conclusion This bespoken, easily adaptable, protective head cap supports postoperative wound healing, and enhances the welfare of monkeys involved in neuroscience research.

Brain perturbation studies allow detailed causal inferences of behavioral and neural processes. Because the combination of brain perturbation methods and neural measurement techniques is inherently challenging, research in humans has predominantly focused on non-invasive, indirect brain perturbations, or neurological lesion studies. Non-human primates have been indispensable as a neurobiological system that is highly similar to humans while simultaneously being more experimentally tractable, allowing visualization of the functional and structural impact of systematic brain perturbation. This review considers the state of the art in non-human primate brain perturbation with a focus on approaches that can be combined with neuroimaging. We consider both non-reversible (lesions) and reversible or temporary perturbations such as electrical, pharmacological, optical, optogenetic, chemogenetic, pathway-selective, and ultrasound based interference methods. Method-specific considerations from the research and development community are offered to facilitate research in this field and support further innovations. We conclude by identifying novel avenues for further research and innovation and by highlighting the clinical translational potential of the methods. ispartof: NEUROIMAGE vol:235 ispartof: location:United States status: published

Summary Theta (3–9 Hz) and gamma (30–100 Hz) oscillations have been observed at different levels along the hierarchy of cortical areas and across a wide set of cognitive tasks. In the visual system, the emergence of both rhythms in primary visual cortex (V1) and mid-level cortical areas V4 has been linked with variations in perceptual reaction times. 1 , 2 , 3 , 4 , 5 Based on analytical methods to infer causality in neural activation patterns, it was concluded that gamma and theta oscillations might both reflect feedforward sensory processing from V1 to V4. 6 , 7 , 8 , 9 , 10 Here, we report on experiments in macaque monkeys in which we experimentally assessed the presence of both oscillations in the neural activity recorded from multi-electrode arrays in V1 and V4 before and after a permanent V1 lesion. With intact cortex, theta and gamma oscillations could be reliably elicited in V1 and V4 when monkeys viewed a visual contour illusion and showed phase-to-amplitude coupling. Laminar analysis in V1 revealed that both theta and gamma oscillations occurred primarily in the supragranular layers, the cortical output compartment of V1. However, there was a clear dissociation between the two rhythms in V4 that became apparent when the major feedforward input to V4 was removed by lesioning V1: although V1 lesioning eliminated V4 theta, it had little effect on V4 gamma power except for delaying its emergence by >100 ms. These findings suggest that theta is more tightly associated with feedforward processing than gamma and pose limits on the proposed role of gamma as a feedforward mechanism.

Natural vision engages a wide range of higher-level regions that integrate visual information over the large-scale brain network. How interareal connectivity reconfigures during the processing of ongoing natural visual scenes and how these dynamic functional changes relate to the underlaying anatomical links between regions is not well understood. Here, we hypothesized that macaque visual brain regions are poly-functional sharing the capacity to change their configuration state depending on the nature of visual input. To address this hypothesis, we reconstructed networks from in-vivo diffusion-weighted imaging (DWI) and functional magnetic resonance imaging (fMRI) data obtained in four alert macaque monkeys viewing naturalistic movie scenes. At first, we characterized network properties and found greater interhemispheric density and greater inter-subject variability in free-viewing networks as compared to structural networks. From the structural connectivity, we then captured modules on which we identified hubs during free-viewing that formed a widespread visuo-saccadic network across frontal (FEF, 46v), parietal (LIP, Tpt), and occipitotemporal modules (MT, V4, TEm), and that excluded primary visual cortex. Inter-subject variability of well-connected hubs reflected subject-specific configurations that largely recruited occipito-parietal and frontal modules. Across the cerebral hemispheres, free-viewing networks showed higher correlations among long-distance brain regions as compared to structural networks. From these findings, we hypothesized that long-distance interareal connectivity could reconfigure depending on the ongoing changes in visual scenes. Testing this hypothesis by applying temporally resolved functional connectivity we observed that many structurally defined areas (such as areas V4, MT/MST and LIP) were poly-functional as they were recruited as hub members of multiple network states that changed during the presentation of scenes containing objects, motion, faces, and actions. We suggest that functional flexibility in macaque macroscale brain networks is required for the efficient interareal communication during active natural vision. To further promote the use of naturalistic free-viewing paradigms and increase the development of macaque neuroimaging resources, we share our datasets in the PRIME-DE consortium.

The liver is the largest organ in the human body and is prone for cancer metastasis. Although the metastatic pattern can differ depending on the cancer type, the liver is the organ to which cancer cells most frequently metastasize for the majority of prevalent malignancies. The liver is unique in several aspects: the vascular structure is highly permeable and has unparalleled dual blood connectivity, and the hepatic tissue microenvironment presents a natural soil for the seeding of disseminated tumor cells. Although 70% of the liver is composed of the parenchymal hepatocytes, the remaining 30% is composed of nonparenchymal cells including Kupffer cells, liver sinusoidal endothelial cells, and hepatic stellate cells. Recent discoveries show that both the parenchymal and the nonparenchymal cells can modulate each step of the hepatic metastatic cascade, including the initial seeding and colonization as well as the decision to undergo dormancy versus outgrowth. Thus, a better understanding of the molecular mechanisms orchestrating the formation of a hospitable hepatic metastatic niche and the identification of the drivers supporting this process is critical for the development of better therapies to stop or at least decrease liver metastasis. The focus of this perspective is on the bidirectional interactions between the disseminated cancer cells and the unique hepatic metastatic niche.

Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest cancers due to its aggressive and metastatic nature. PDA is characterized by a rich tumor stroma with abundant macrophages, fibroblasts and collagen deposition that can represent up to 90% of the tumor mass. Activation of the tyrosine kinase receptor AXL and expression of its ligand growth arrest-specific protein 6 (Gas6) correlate with a poor prognosis and increased metastasis in pancreatic cancer patients. Gas6 is a multifunctional protein that can be secreted by several cell types and regulates multiple processes, including cancer cell plasticity, angiogenesis and immune cell functions. However, the role of Gas6 in pancreatic cancer metastasis has not been fully investigated. In these studies we find that, in pancreatic tumors, Gas6 is mainly produced by tumor associated macrophages (TAMs) and cancer associated fibroblasts (CAFs) and that pharmacological blockade of Gas6 partially reverses epithelial-to-mesenchymal transition (EMT) of tumor cells and supports NK cell activation, thereby inhibiting pancreatic cancer metastasis. Our data suggest that Gas6 simultaneously acts on both the tumor cells and the NK cells to support pancreatic cancer metastasis. This study supports the rationale for targeting Gas6 in pancreatic cancer and use NK cells as a potential biomarker for response to anti-Gas6 therapy.