Abstract Immunosuppression in glioblastoma is a powerful impediment to effective immunotherapy. We used maximal tumor sampling and genome-wide coexpression analysis of RNA sequencing, on 69 samples from 8 patients, to identify a module of 270 genes marked by LRRC25, determined by top fidelity score, that covaries with many immunosuppressive molecules. An immunosuppressive module was independently derived from an expanded cohort of 319 samples from 28 GBM also marked by LRRC25 by top fidelity score. Within the gene module, we observed strong co-variation of markers for monocytes, neutrophils, and NK cells with LRRC25. In published single-cell RNA-seq from glioma, we determined that LRRC25 is expressed by neutrophils and myeloid cells while absent from NK-cells. LRRC25 is also constitutively expressed in monocytes and neutrophils while being upregulated by microglia and DCs in the tumor core and necrotic regions. In addition to covariation with multiple cell-type specific markers we also discovered that LRRC25 expression is tightly associated with several members of the TYROBP causal signaling network in microglia. Thus, LRRC25 may play a novel role in TYROBP signaling which may, in turn, represent a novel mechanism of NK cell and neutrophil immunosuppression in glioma. We confirmed LRRC25 RNA expression in patient samples by RNAscope co-stained with an anti-CD163 antibody indicating myeloid expression, as well as tumor cell expression determined by histopathology. Although LRRC25 is known to mediate IFN and NFKb signaling in myeloid cells in-vitro, its function in glioma remains understudied. While significant efforts have focused on mechanisms of myeloid to T-cell immunosuppression, less focus has been given to mechanisms of myeloid to NK-cell and neutrophil immunosuppression, despite the fact they exist at levels comparable to T-cells in the tumor microenvironment. This work will be followed up with antibody validation as well as genetic manipulation of LRRC25 in-vitro and in-vivo.
<div>AbstractPurpose:<p>Patients with central nervous system (CNS) tumors are typically treated with radiotherapy, but this is not curative and results in the upregulation of phosphorylated STAT3 (p-STAT3), which drives invasion, angiogenesis, and immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3 and whole-brain radiotherapy (WBRT) in a murine model of glioma.</p>Experimental Design:<p>C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT, and treatment with WP1066, a blood–brain barrier–penetrant inhibitor of the STAT3 pathway, or the two in combination. The role of the immune system was evaluated using tumor rechallenge strategies, immune-incompetent backgrounds, immunofluorescence, immune phenotyping of tumor-infiltrating immune cells (via flow cytometry), and NanoString gene expression analysis of 770 immune-related genes from immune cells, including those directly isolated from the tumor microenvironment.</p>Results:<p>The combination of WP1066 and WBRT resulted in long-term survivors and enhanced median survival time relative to monotherapy in the GL261 glioma model (combination vs. control <i>P</i> < 0.0001). Immunologic memory appeared to be induced, because mice were protected during subsequent tumor rechallenge. The therapeutic effect of the combination was completely lost in immune-incompetent animals. NanoString analysis and immunofluorescence revealed immunologic reprograming in the CNS tumor microenvironment specifically affecting dendritic cell antigen presentation and T-cell effector functions.</p>Conclusions:<p>This study indicates that the combination of STAT3 inhibition and WBRT enhances the therapeutic effect against gliomas in the CNS by inducing dendritic cell and T-cell interactions in the CNS tumor.</p></div>
Abstract We have recently demonstrated that natural killer (NK) cells eradicate galectin-1 (gal-1)-deficient glioma (Baker et al, Cancer Research. 2014. Sep 15;74(18):5079-90). Here we present our progress towards understanding the cellular mechanism(s) by which NK cells eradicate such tumors. We demonstrate that monocytic Gr-1+/CD11b+ myeloid cells are required to stimulate NK-mediated lysis of gal-1-deficient glioma. Immunodepletion of Gr-1+ cells in RAG1-/- mice permits lethal gal-1-deficient glioma formation despite the presence of NK cells, demonstrating that Gr-1+ cells (i.e. myeloid cells) are necessary for NK-mediated tumor rejection in vivo. We also demonstrate that the effect of glioma-derived gal-1 is to conceal glioma cells from recognition by NK cells by inhibiting myeloid-NK cell crosstalk. In vitro experiments reveal that gal-1-deficient glioma cells stimulate cross-activation between NK cells and monocytic Gr-1+/CD11b+ myeloid cells, causing the myeloid cells to morph into a phenotypically distinct cell population reminiscent of macrophages or dendritic cells. Cell-killing assays show that monocytic Gr-1+/CD11b+ myeloid cells significantly enhance NK-mediated glioma cell lysis in vitro. Recombinant mouse gal-1 protein inhibits the myeloid cell enhancement of NK-mediated tumor lysis, but fails to suppress intrinsic NK-mediated tumor lysis. This result strongly suggests that the role of glioma-derived gal-1 is to suppress the ability of monocytic myeloid cells to stimulate cytotoxic potential in NK cells. Further in vivo experiments also reveal that RAG1-/- mice bearing gal-1-deficient glioma have 7-fold more Gr-1+/CD11b+ myeloid cells present within the tumor microenvironment 48hrs after tumor implantation compared to gal-1-expressing tumors. Together our data show that glioma-derived gal-1 acts to potently suppress anti-glioma NK immune surveillance through a tripartite mechanism involving: (1) the inhibition of myeloid cell recruitment into the brain tumor microenvironment, (2) the suppression of enhanced NK-mediated glioma lysis stimulated by monocytic myeloid cells, and (3) an increased resistance to NK-mediated glioma cell lysis. We now aim to identify the molecular factors used by gal-1-deficient glioma cells to stimulate monocytic myeloid cell activation and the factors that myeloid cells use to in turn amplify NK cell cytotoxicity. Citation Format: Gregory J. Baker, Peter Chockley, Daniel Zamler, Viveka Nand Yadav, Maria G. Castro, Pedro R. Lowenstein. Monocytic Gr-1+/CD11b+ myeloid cells are necessary for natural killer cells to eradicate glioma and are inhibited by tumor-derived galectin-1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 452. doi:10.1158/1538-7445.AM2015-452
Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)–mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4–mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.
Intra-tumoral heterogeneity is a hallmark of glioblastoma that challenges treatment efficacy. However, the mechanisms that set up tumor heterogeneity and tumor cell migration remain poorly understood. Herein, we present a comprehensive spatiotemporal study that aligns distinctive intra-tumoral histopathological structures, oncostreams, with dynamic properties and a specific, actionable, spatial transcriptomic signature. Oncostreams are dynamic multicellular fascicles of spindle-like and aligned cells with mesenchymal properties, detected using ex vivo explants and in vivo intravital imaging. Their density correlates with tumor aggressiveness in genetically engineered mouse glioma models, and high grade human gliomas. Oncostreams facilitate the intra-tumoral distribution of tumoral and non-tumoral cells, and potentially the collective invasion of the normal brain. These fascicles are defined by a specific molecular signature that regulates their organization and function. Oncostreams structure and function depend on overexpression of COL1A1. Col1a1 is a central gene in the dynamic organization of glioma mesenchymal transformation, and a powerful regulator of glioma malignant behavior. Inhibition of Col1a1 eliminates oncostreams, reprograms the malignant histopathological phenotype, reduces expression of the mesenchymal associated genes, induces changes in the tumor microenvironment and prolongs animal survival. Oncostreams represent a pathological marker of potential value for diagnosis, prognosis, and treatment.
