ABSTRACT
The immune fraction of the tumor microenvironment has been proven to play a fundamental role in glioblastoma progression and therapeutic response. Here, we present a detailed magnetic-bead-enrichment-based protocol to isolate and analyze the composition of this fraction from mouse brain tumors. The protocol is optimized to achieve high yields of viable immune cells. We also detail characterization of the immune subtypes by FACS analysis. Our procedure is applicable for either lentiviral-induced tumors or transplant models in syngeneic immunocompetent mice. For complete details on the use and execution of this protocol, please refer to Magod et al. (2021).
Subject(s)
Brain Neoplasms/immunology , Tumor Microenvironment , Animals , Brain Neoplasms/pathology , Disease Models, Animal , Flow Cytometry , MiceABSTRACT
Recent multi-omics studies show different immune tumor microenvironment (TME) compositions in glioblastoma (GBM). However, temporal comprehensive knowledge of the TME from initiation of the disease remains sparse. We use Cre recombinase (Cre)-inducible lentiviral murine GBM models to compare the cellular evolution of the immune TME in tumors initiated from different oncogenic drivers. We show that neutrophils infiltrate early during tumor progression primarily in the mesenchymal GBM model. Depleting neutrophils in vivo at the onset of disease accelerates tumor growth and reduces the median overall survival time of mice. We show that, as a tumor progresses, bone marrow-derived neutrophils are skewed toward a phenotype associated with pro-tumorigenic processes. Our findings suggest that GBM can remotely regulate systemic myeloid differentiation in the bone marrow to generate neutrophils pre-committed to a tumor-supportive phenotype. This work reveals plasticity in the systemic immune host microenvironment, suggesting an additional point of intervention in GBM treatment.
Subject(s)
Bone Marrow/pathology , Brain Neoplasms/pathology , Carcinogenesis/pathology , Glioma/pathology , Neutrophils/pathology , Tumor Microenvironment , Animals , Brain Neoplasms/blood supply , Brain Neoplasms/drug therapy , Cell Line, Tumor , Cytotoxicity, Immunologic , Disease Models, Animal , Disease Progression , Female , Glioma/blood supply , Glioma/drug therapy , Humans , Immunosuppression Therapy , Integrases/metabolism , Mice, Inbred C57BL , Mutation/genetics , Neoplasm Staging , Neovascularization, Pathologic/pathology , Survival AnalysisABSTRACT
Glioblastoma is considered one of the most aggressive malignancies in adult and pediatric patients. Despite decades of research no curative treatment is available and it thus remains associated with a very dismal prognosis. Although recent pre-clinical and clinical studies have demonstrated the feasibility of chimeric antigen receptors (CAR) T cell immunotherapeutic approach in glioblastoma, tumor heterogeneity and antigen loss remain among one of the most important challenges to be addressed. In this study, we identify p32/gC1qR/HABP/C1qBP to be specifically expressed on the surface of glioma cells, making it a suitable tumor associated antigen for redirected CAR T cell therapy. We generate p32 CAR T cells and find them to recognize and specifically eliminate p32 expressing glioma cells and tumor derived endothelial cells in vitro and to control tumor growth in orthotopic syngeneic and xenograft mouse models. Thus, p32 CAR T cells may serve as a therapeutic option for glioblastoma patients.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Antineoplastic Agents/pharmacology , Glioma/immunology , Glioma/therapy , T-Lymphocytes/immunology , Aged , Animals , Antigens, Neoplasm/immunology , Brain Neoplasms , Carrier Proteins/metabolism , Cell Line, Tumor , Female , Glioblastoma/genetics , Glioblastoma/pathology , Glioma/genetics , Glioma/metabolism , Humans , Immunotherapy, Adoptive , Male , Mice , Mice, Inbred C57BL , Mice, Nude , Mitochondrial Proteins/metabolism , Receptors, Antigen, T-Cell/metabolism , Receptors, Chimeric Antigen/immunology , Serine Endopeptidases/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Tumor-selective drug conjugates can potentially improve the prognosis for patients affected by glioblastoma (GBM) - the most common and malignant type of brain cancer with no effective cure. Here we evaluated a novel tumor penetrating peptide that targets cell surface p32, LinTT1 (AKRGARSTA), as a GBM targeting ligand for systemically-administered nanoparticles. LinTT1-functionalization increased tumor homing of iron oxide nanoworms (NWs) across a panel of five GBM models ranging from infiltratively-disseminating to angiogenic phenotypes. LinTT1-NWs homed to CD31-positive tumor blood vessels, including to transdifferentiated endothelial cells, and showed co-localization with tumor macrophages and lymphatic vessels. LinTT1 functionalization also resulted in increased GBM delivery of other types of systemically-administered nanoparticles: silver nanoparticles and albumin-paclitaxel nanoparticles. Finally, LinTT1-guided proapoptotic NWs exerted strong anti-glioma activity in two models of GBM, including doubling the lifespan of the mice in an aggressive orthotopic stem cell-like GBM that recapitulates the histological hallmarks of human GBM. Our study suggests that LinTT1 targeting strategy can be used to increase GBM uptake of systemic nanoparticles for improved imaging and therapy.
Subject(s)
Brain Neoplasms/drug therapy , Drug Delivery Systems , Glioblastoma/drug therapy , Nanoparticles , Peptides/administration & dosage , Albumins/administration & dosage , Albumins/pharmacokinetics , Animals , Cell Line, Tumor , Endothelial Cells/metabolism , Female , Ferric Compounds/chemistry , Glioblastoma/pathology , Humans , Male , Metal Nanoparticles , Mice , Mice, Inbred C57BL , Mice, Nude , Paclitaxel/administration & dosage , Paclitaxel/pharmacokinetics , Peptides/chemistry , Silver/chemistry , Xenograft Model Antitumor AssaysABSTRACT
The reported new anilinoquinazoline-icosahedral borane hybrids have been evaluated as glioma targeting for potential use in cancer therapy. Their anti-glioma activity depends on hybrids' lipophilicity; the most powerful compound against glioma cells, a 1,7-closo-derivative, displayed at least 3.3â times higher activity than the parent drug erlotinib. According to the cytotoxic effects on normal glia cells, the hybrids were selective for epidermal growth factor receptor (EGFR)-overexpressed tumor cells. These boron carriers could be used to enrich glioma cancer cells with boron for cancer therapy.
Subject(s)
Boranes/chemistry , Protein Kinase Inhibitors/chemistry , Thiazoles/chemistry , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , ErbB Receptors/metabolism , Glioma/metabolism , Glioma/pathology , Humans , Hydrophobic and Hydrophilic Interactions , Inhibitory Concentration 50 , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/toxicityABSTRACT
Cancer chemoprevention involves prevention/delay/reverse of the carcinogenic process through administration of cancer chemopreventive agents (CCA). Compounds which are able to induce detoxification-enzymes, especially monofunctional phase II enzymes, have become in excellent approaches for new CCA. Herein, we report the synthesis of new furoxanyl chalcone-like hybrid compounds as CCA. In vitro studies showed that phenylfuroxanyl derivatives 6 and 9 displayed the best activities being 9 the greatest monofunctional-inducer. Additionally, compounds were non-mutagenic against TA98 Salmonella typhimurium strain (Ames test) and could be used in the prevention of the progression of pre-malignant lesions for their cytotoxic activity against tumoral cells. In vivo proof of concept showed increment on phase II-enzymes activities in liver, colon and mammary gland having derivative 9 the best induction profiles. We probed Nrf2 nuclear translocation is operative for both compounds allowing to exert protective effects via expression of downstream phase-II enzymes.