Coordinated single-cell tumor microenvironment dynamics reinforce pancreatic cancer subtype.

Bulk analyses of pancreatic ductal adenocarcinoma (PDAC) samples are complicated by the tumor microenvironment (TME), i.e. signals from fibroblasts, endocrine, exocrine, and immune cells. Despite this, we and others have established tumor and stroma subtypes with prognostic significance. However, understanding of underlying signals driving distinct immune and stromal landscapes is still incomplete. Here we integrate 92 single cell RNA-seq samples from seven independent studies to build a reproducible PDAC atlas with a focus on tumor-TME interdependence. Patients with activated stroma are synonymous with higher myofibroblastic and immunogenic fibroblasts, and furthermore show increased M2-like macrophages and regulatory T-cells. Contrastingly, patients with 'normal' stroma show M1-like recruitment, elevated effector and exhausted T-cells. To aid interoperability of future studies, we provide a pretrained cell type classifier and an atlas of subtype-based signaling factors that we also validate in mouse data. Ultimately, this work leverages the heterogeneity among single-cell studies to create a comprehensive view of the orchestra of signaling interactions governing PDAC.

A Novel Class of Ribosome Modulating Agents Exploits Cancer Ribosome Heterogeneity to Selectively Target the CMS2 Subtype of Colorectal Cancer.

Ribosomes in cancer cells accumulate numerous patient-specific structural and functional modifications that facilitate tumor progression by modifying protein translation. We have taken a unique synthetic chemistry approach to generate novel macrolides, Ribosome modulating agents (RMA), that are proposed to act distal to catalytic sites and exploit cancer ribosome heterogeneity. The RMA ZKN-157 shows two levels of selectivity: (i) selective translation inhibition of a subset of proteins enriched for components of the ribosome and protein translation machinery that are upregulated by MYC; and (ii) selective inhibition of proliferation of a subset of colorectal cancer cell lines. Mechanistically, the selective ribosome targeting in sensitive cells triggered cell-cycle arrest and apoptosis. Consequently, in colorectal cancer, sensitivity to ZKN-157 in cell lines and patient-derived organoids was restricted to the consensus molecular subtype 2 (CMS2) subtype that is distinguished by high MYC and WNT pathway activity. ZKN-157 showed efficacy as single agent and, the potency and efficacy of ZKN-157 synergized with clinically approved DNA-intercalating agents which have previously been shown to inhibit ribogenesis as well. ZKN-157 thus represents a new class of ribosome modulators that display cancer selectivity through specific ribosome inhibition in the CMS2 subtype of colorectal cancer potentially targeting MYC-driven addiction to high protein translation. Significance: This study demonstrates that ribosome heterogeneity in cancer can be exploited to develop selective ribogenesis inhibitors. The colorectal cancer CMS2 subtype, with a high unmet need for therapeutics, shows vulnerability to our novel selective ribosome modulator. The mechanism suggests that other cancer subtypes with high MYC activation could also be targeted.

A distinct microglial subset at the tumor-stroma interface of glioma.

The characterization of the tumor microenvironment (TME) in high grade gliomas (HGG) has generated significant interest in an effort to understand how neoplastic lesions in the central nervous system (CNS) are supported and to devise novel therapeutic targets. The TME of the CNS contains unique and specialized cells, including the resident myeloid cells, microglia. Myeloid involvement in HGG, such as glioblastoma, is associated with poor outcomes. Glioma-associated microglia and infiltrating monocytes/macrophages (GAM) accumulate within the neoplastic lesion where they facilitate tumor growth and drive immunosuppression. However, it has been difficult to differentiate whether microglia and macrophages have similar or distinct roles in pathology, and if the spatial organization of these cells informs outcomes. Here, we characterize the tumor-stroma border and identify peritumoral GAM (PGAM) as a unique subpopulation of GAM. Using data mining and analyses of samples derived from both murine and human sources we show that PGAM exhibit a pro-inflammatory and chemotactic phenotype that is associated with peripheral monocyte recruitment, and decreased overall survival. PGAM act as a unique subset of GAM at the tumor-stroma interface. We define a novel gene signature to identify these cells and suggest that PGAM constitute a cellular target of the TME.

A Rigorous Quantitative Approach to Analyzing Phagocytosis Assays.

Studying monocytic cells in isolated systems in vitro contributes significantly to the understanding of innate immune physiology. Functional assays produce read outs which can be used to measure responses to selected stimuli, such as pathogen exposure, antigen loading, and cytokine stimulation. Integration of these results with high quality in vivo models allows for the development of therapeutics which target these cell populations. Current methodologies to quantify phagocytic function of monocytic cells in vitro either measure phagocytic activity of individual cells (average number of beads or particles/cell), or a population outcome (% cells that contain phagocytosed material). Here we address technical challenges and shortcomings of these methods and present a protocol for collecting and analyzing data derived from a functional assay which measures phagocytic activity of macrophage and macrophage-like cells. We apply this method to two different experimental conditions, and compare to existing work flows. We also provide an online tool for users to upload and analyze data using this method.

BSSE: An open-source image processing tool for miniaturized microscopy.

Single-photon-excitation-based miniaturized microscope, or miniscope, has recently emerged as a powerful tool for imaging neural ensemble activities in freely moving animals. In the meanwhile, this highly flexible and implantable technology promises great potential for studying a broad range of cells, tissues and organs. To date, however, applications have been largely limited by the properties of the imaging modality. It is therefore highly desirable for a method generally applicable for processing miniscopy images, enabling and extending the applications to diverse anatomical and functional traits, spanning various cell types in the brain and other organs. We report an image processing approach, termed BSSE, for background suppression and signal enhancement for miniscope image processing. The BSSE method provides a simple, automatic solution to the intrinsic challenges of overlapping signals, high background and artifacts in miniscopy images. We validated the method by imaging synthetic structures and various biological samples of brain, tumor, and kidney tissues. The work represents a generally applicable tool for miniscopy technology, suggesting broader applications of the miniaturized, implantable and flexible technology for biomedical research.

Pifithrin-µ modulates microglial activation and promotes histological recovery following spinal cord injury.

BACKGROUND: Treatments immediately after spinal cord injury (SCI) are anticipated to decrease neuronal death, disruption of neuronal connections, demyelination, and inflammation, and to improve repair and functional recovery. Currently, little can be done to modify the acute phase, which extends to the first 48 hours post-injury. Efforts to intervene have focused on the subsequent phases - secondary (days to weeks) and chronic (months to years) - to both promote healing, prevent further damage, and support patients suffering from SCI. METHODS: We used a contusion model of SCI in female mice, and delivered a small molecule reagent during the early phase of injury. Histological and behavioral outcomes were assessed and compared. RESULTS: We find that the reagent Pifithrin-µ (PFT-µ) acts early and directly on microglia in vitro, attenuating their activation. When administered during the acute phase of SCI, PFT-µ resulted in reduced lesion size during the initial inflammatory phase, and reduced the numbers of pro-inflammatory microglia and macrophages. Treatment with PFT-µ during the early stage of injury maintained a stable anti-inflammatory environment. CONCLUSIONS: Our results indicate that a small molecule reagent PFT-µ has sustained immunomodulatory effects following a single dose after injury.

Expression of neuropilin-1 is linked to glioma associated microglia and macrophages and correlates with unfavorable prognosis in high grade gliomas.

High grade gliomas, including glioblastoma (GB), are devastating malignancies with very poor prognosis. Over the course of the last decade, there has been a failure to develop new treatments for GB. Reasons for this failure include the lack of validation of novel molecular targets, which are often characterized in animal models and directly transposed to human trials. Here we build on our previous findings, which describe how the multi-functional co-receptor Neuropilin-1 (NRP1) signals through glioma associated microglia/macrophages (GAMS) to promote murine glioma, and investigate NRP1 expression in human glioma. Clinical and gene expression data were obtained via The Cancer Genome Atlas (TCGA), and analyzed using R statistical software. Additionally, CIBERSORT in silico deconvolution was used to determine fractions of immune cell sub-populations within the gene expression datasets. We find that NRP1 expression is correlated with poor prognosis, glioma grade, and associates with the mesenchymal GB subtype. In human GB, NRP1 expression is highly correlated with markers of monocytes/macrophages, as well as genes that contribute to the pro-tumorigenic phenotype of these cells.

Contributions of immune cell populations in the maintenance, progression, and therapeutic modalities of glioma.

Immunotherapies are becoming a promising strategy for malignant disease. Selectively directing host immune responses to target cancerous tissue is a milestone of human health care. The roles of the innate and adaptive immune systems in both cancer progression and elimination are now being realized. Defining the immune cell environment and identifying the contributions of each sub-population of these cells has lead to an understanding of the immunotherapeutic processes, and demonstrated the potential of the immune system to drive cancer shrinkage and sustained immunity against disease. Poorly treated diseases, such as high-grade glioma, suffer from lack of therapeutic efficacy and rapid progression. Immunotherapeutic success in other solid malignancies, such as melanoma, now provides the principals for which this treatment paradigm can be adapted for primary brain cancers. The central nervous system is complex, and relative contributions of immune sub-populations to high grade glioma progression are not fully characterized. Here, we summarize recent research in both animal and humans which add to the knowledge base of how innate and adaptive immune cells contribute to glioma progression, and outline work which has demonstrated their potential to elicit anti-tumorigenic responses. Additionally, we highlight Neuropilin 1, a cell surface receptor protein, describe its signaling functions in the context of immunity, and point to its potential to slow glioma progression.

Deletion of Neuropilin 1 from Microglia or Bone Marrow-Derived Macrophages Slows Glioma Progression.

Glioma-associated microglia and macrophages (GAM), which infiltrate high-grade gilomas, constitute a major cellular component of these lesions. GAM behavior is influenced by tumor-derived cytokines that suppress initial antitumorigenic properties, causing them to support tumor growth and to convert and suppress adaptive immune responses to the tumor. Mice that lack the transmembrane receptor neuropilin-1 (Nrp1), which modulates GAM immune polarization, exhibit a decrease in glioma volumes and neoangiogenesis and an increase in antitumorigenic GAM infiltrate. Here we show that replacing the peripheral macrophage populations of wild-type mice with Nrp1-depleted bone marrow-derived macrophages (BMDM) confers resistance to the development of glioma. This resistance occurred in a similar fashion seen in mice in which all macrophages lacked Nrp1 expression. Tumors had decreased volumes, decreased vascularity, increased CTL infiltrate, and Nrp1-depleted BMDM adopted a more antitumorigenic phenotype relative to wild-type GAMs within the tumors. Mice with Nrp1-deficient microglia and wild-type peripheral macrophages showed resistance to glioma development and had higher microglial infiltrate than mice with wild-type GAMs. Our findings show how manipulating Nrp1 in either peripheral macrophages or microglia reprograms their phenotype and their pathogenic roles in tumor neovascularization and immunosuppression.Significance: This study highlights the proangiogenic receptor neuropilin 1 in macrophages and microglial cells in gliomas as a pivotal modifier of tumor neovascularization and immunosuppression, strengthening emerging evidence of the functional coordination of these two fundamental traits of cancer. Cancer Res; 78(3); 685-94. ©2017 AACR.