Immune landscape of isocitrate dehydrogenase-stratified primary and recurrent human gliomas.

BACKGROUND: Human gliomas are classified using isocitrate dehydrogenase (IDH) status as a prognosticator; however, the influence of genetic differences and treatment effects on ensuing immunity remains unclear. METHODS: In this study, we used sequential single-cell transcriptomics on 144,678 and spectral cytometry on over two million immune cells encompassing 48 human gliomas to decipher their immune landscape. RESULTS: We identified 22 distinct immune cell types that contribute to glioma immunity. Specifically, brain-resident microglia (MG) were reduced with a concomitant increase in CD8+ T lymphocytes during glioma recurrence independent of IDH status. In contrast, IDH-wild-type-associated patterns, such as an abundance of antigen-presenting cell-like MG and cytotoxic CD8+ T cells, were observed. Beyond elucidating the differences in IDH, relapse, and treatment-associated immunity, we discovered novel inflammatory MG subpopulations expressing granulysin, a cytotoxic peptide, which is otherwise expressed in lymphocytes only. Furthermore, we provide a robust genomic framework for defining macrophage polarization beyond M1/M2 paradigm and reference signatures of glioma-specific tumor immune microenvironment (termed Glio-TIME-36) for deconvoluting transcriptomic datasets. CONCLUSIONS: This study provides advanced optics of the human pan-glioma immune contexture as a valuable guide for translational and clinical applications.

Triggering receptor expressed on myeloid cells 2 (TREM2) regulates phagocytosis in glioblastoma.

BACKGROUND: Glioblastomas (GBMs) are central nervous system tumors that resist standard-of-care interventions and even immune checkpoint blockade. Myeloid cells in the tumor microenvironment can contribute to GBM progression; therefore, emerging immunotherapeutic approaches include reprogramming these cells to achieve desirable antitumor activity. Triggering receptor expressed on myeloid cells 2 (TREM2) is a myeloid signaling regulator that has been implicated in a variety of cancers and neurological diseases with contrasting functions, but its role in GBM immunopathology and progression is still under investigation. METHODS: Our reverse translational investigations leveraged single-cell RNA sequencing and cytometry of human gliomas to characterize TREM2 expression across myeloid subpopulations. Using 2 distinct murine glioma models, we examined the role of Trem2 on tumor progression and immune modulation of myeloid cells. Furthermore, we designed a method of tracking phagocytosis of glioma cells in vivo and employed in vitro assays to mechanistically understand the influence of TREM2 signaling on tumor uptake. RESULTS: We discovered that TREM2 expression does not correlate with immunosuppressive pathways, but rather showed strong a positive association with the canonical phagocytosis markers lysozyme (LYZ) and macrophage scavenger receptor (CD163) in gliomas. While Trem2 deficiency was found to be dispensable for gliomagenesis, Trem2+ myeloid cells display enhanced tumor uptake compared to Trem2- cells. Mechanistically, we demonstrate that TREM2 mediates phagocytosis via Syk signaling. CONCLUSIONS: These results indicate that TREM2 is not associated with immunosuppression in gliomas. Instead, TREM2 is an important regulator of phagocytosis that may be exploited as a potential therapeutic strategy for brain tumors.

Integrated proteogenomic characterization of glioblastoma evolution.

The evolutionary trajectory of glioblastoma (GBM) is a multifaceted biological process that extends beyond genetic alterations alone. Here, we perform an integrative proteogenomic analysis of 123 longitudinal glioblastoma pairs and identify a highly proliferative cellular state at diagnosis and replacement by activation of neuronal transition and synaptogenic pathways in recurrent tumors. Proteomic and phosphoproteomic analyses reveal that the molecular transition to neuronal state at recurrence is marked by post-translational activation of the wingless-related integration site (WNT)/ planar cell polarity (PCP) signaling pathway and BRAF protein kinase. Consistently, multi-omic analysis of patient-derived xenograft (PDX) models mirror similar patterns of evolutionary trajectory. Inhibition of B-raf proto-oncogene (BRAF) kinase impairs both neuronal transition and migration capability of recurrent tumor cells, phenotypic hallmarks of post-therapy progression. Combinatorial treatment of temozolomide (TMZ) with BRAF inhibitor, vemurafenib, significantly extends the survival of PDX models. This study provides comprehensive insights into the biological mechanisms of glioblastoma evolution and treatment resistance, highlighting promising therapeutic strategies for clinical intervention.

The Epigenetic Evolution of Glioma Is Determined by the IDH1 Mutation Status and Treatment Regimen.

Tumor adaptation or selection is thought to underlie therapy resistance in glioma. To investigate longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 132 matched initial and recurrent tumors from patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) glioma. IDHwt gliomas showed a stable epigenome over time with relatively low levels of global methylation. The epigenome of IDHmut gliomas showed initial high levels of genome-wide DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. Integration of epigenomics, gene expression, and functional genomics identified HOXD13 as a master regulator of IDHmut astrocytoma evolution. Furthermore, relapse of IDHmut tumors was accompanied by histologic progression that was associated with survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment varied between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neoangiogenesis and T-cell infiltration upon treatment of IDHmut gliomas. This study provides one of the largest cohorts of paired longitudinal glioma samples with epigenomic, transcriptomic, and genomic profiling and suggests that treatment of IDHmut glioma is associated with epigenomic evolution toward an IDHwt-like phenotype. SIGNIFICANCE: Standard treatments are related to loss of DNA methylation in IDHmut glioma, resulting in epigenetic activation of genes associated with tumor progression and alterations in the microenvironment that resemble treatment-naïve IDHwt glioma.

Integrative multi-omics networks identify PKCδ and DNA-PK as master kinases of glioblastoma subtypes and guide targeted cancer therapy.

Despite producing a panoply of potential cancer-specific targets, the proteogenomic characterization of human tumors has yet to demonstrate value for precision cancer medicine. Integrative multi-omics using a machine-learning network identified master kinases responsible for effecting phenotypic hallmarks of functional glioblastoma subtypes. In subtype-matched patient-derived models, we validated PKCδ and DNA-PK as master kinases of glycolytic/plurimetabolic and proliferative/progenitor subtypes, respectively, and qualified the kinases as potent and actionable glioblastoma subtype-specific therapeutic targets. Glioblastoma subtypes were associated with clinical and radiomics features, orthogonally validated by proteomics, phospho-proteomics, metabolomics, lipidomics and acetylomics analyses, and recapitulated in pediatric glioma, breast and lung squamous cell carcinoma, including subtype specificity of PKCδ and DNA-PK activity. We developed a probabilistic classification tool that performs optimally with RNA from frozen and paraffin-embedded tissues, which can be used to evaluate the association of therapeutic response with glioblastoma subtypes and to inform patient selection in prospective clinical trials.

The AKT1E17K Allele Promotes Breast Cancer in Mice.

The gain-of-function mutation in the pleckstrin homology domain of AKT1 (AKT1E17K) occurs in lung and breast cancer. Through the use of human cellular models and of a AKT1E17K transgenic Cre-inducible murine strain (R26-AKT1E17K mice), we have demonstrated that AKT1E17K is a bona fide oncogene for lung epithelial cells. However, the role of AKT1E17K in breast cancer remains to be determined. Here, we report the generation and the characterization of a MMTV-CRE; R26-AKT1E17K mouse strain that expresses the mutant AKT1E17K allele in the mammary epithelium. We observed that AKT1E17K stimulates the development of mammary tumors classified as ductal adenocarcinoma of medium-high grade and presented a variety of proliferative alterations classified as adenosis with low-to-high grade dysplasia in the mammary epithelium. A subsequent immunohistochemical characterization suggested they were PR-/HER2-/ER+, basal-like and CK8-/CK10-/CK5+/CK14+. We also observed that, in parallel with an increased proliferation rate, tumors expressing mutant AKT1E17K presented an activation of the GSK3/cyclin D1 pathway in the mammary epithelium and cluster significantly with the human basal-like tumors. In conclusion, we demonstrate AKT1E17K is a bona fide oncogene that can initiate tumors at high efficiency in murine mammary epithelium in vivo.

Glioma progression is shaped by genetic evolution and microenvironment interactions.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.

Effect of D-Mannitol on the Microstructure and Rheology of Non-Aqueous Carbopol Microgels.

D-mannitol is a common polyol that is used as additive in pharmaceutical and personal care product formulations. We investigated its effect on the microstructure and rheology of novel non-aqueous Carbopol dispersions employing traditional and time-resolved rheological analysis. We considered two types of sample, (i) fresh (i.e., mannitol completely dissolved in solution) and aged (i.e., visible in crystalline form). The analysis of the intracycle rheological transitions that were observed for different samples revealed that, when completely dissolved in solution, mannitol does not alter the rheological behaviour of the Carbopol dispersions. This highlights that the chemical similarity of the additive with the molecules of the surrounding solvent allows preserving the swollen dimension and interparticle interactions of the Carbopol molecules. Conversely, when crystals are present, a hierarchical structure forms, consisting of a small dispersed phase (Carbopol) agglomerated around a big dispersed phase (crystals). In keeping with this microstructural picture, as the concentration of Carbopol reduces, the local dynamics of the crystals gradually start to control the integrity of the microstructure. Rheologically, this results in a higher elasticity of the suspensions at infinitesimal deformations, but a fragile yielding process at intermediate strains.

Pathway-based classification of glioblastoma uncovers a mitochondrial subtype with therapeutic vulnerabilities.

The transcriptomic classification of glioblastoma (GBM) has failed to predict survival and therapeutic vulnerabilities. A computational approach for unbiased identification of core biological traits of single cells and bulk tumors uncovered four tumor cell states and GBM subtypes distributed along neurodevelopmental and metabolic axes, classified as proliferative/progenitor, neuronal, mitochondrial and glycolytic/plurimetabolic. Each subtype was enriched with biologically coherent multiomic features. Mitochondrial GBM was associated with the most favorable clinical outcome. It relied exclusively on oxidative phosphorylation for energy production, whereas the glycolytic/plurimetabolic subtype was sustained by aerobic glycolysis and amino acid and lipid metabolism. Deletion of the glucose-proton symporter SLC45A1 was the truncal alteration most significantly associated with mitochondrial GBM, and the reintroduction of SLC45A1 in mitochondrial glioma cells induced acidification and loss of fitness. Mitochondrial, but not glycolytic/plurimetabolic, GBM exhibited marked vulnerability to inhibitors of oxidative phosphorylation. The pathway-based classification of GBM informs survival and enables precision targeting of cancer metabolism.

Investigation of the swollen state of Carbopol molecules in non-aqueous solvents through rheological characterization.

We explore how different types of solvent influence the rheological properties of non-aqueous Carbopol dispersions from the dilute to the jammed state. In novel non-aqueous formulations, polar solvents are used more and more frequently, because they can form Carbopol microgels without the need of any neutralizing agents. However, the swelling behaviour of Carbopol molecules in the absence of water, when ionic forces are weak, is still poorly understood. To this end, we study the swelling behaviour of Carbopol 974P NF in different polar solvents, i.e. glycerol, PEG400 and mixtures of the two solvents, by mapping the rheological behaviour of Carbopol suspensions from very dilute to highly concentrated conditions. The rheological study reveals that the onset of the jamming transition occurs at different critical polymer concentrations depending on the solvents used. Nevertheless, once the jammed state is reached, both elastic and yielding behaviours are scalable with the particle volume fraction. These results suggest that the type of solvent influences the final volume of the single Carbopol particles but does not alter the interactions between the particles. The final radius of the swollen particles is estimated from shear rheology measurements in dilute conditions, showing a decrease of the final swelling ratio of Carbopol molecules of almost 50% for PEG400 solutions, a result that confirms the shift to higher values of the critical jamming concentration obtained from linear viscoelasticity for the same solutions.