ABSTRACT
Radiation therapy is a commonly used treatment modality for cancer. Although effective in providing local tumor control, radiation causes oxidative stress, inflammation, immunomodulatory and mitogenic cytokine production, extracellular matrix production, and premature senescence in lung parenchyma. The senescence associated secretory phenotype (SASP) can promote inflammation and stimulate alterations in the surrounding tissue. Therefore, we hypothesized that radiation-induced senescent parenchymal cells in irradiated lung would enhance tumor growth. Using a murine syngeneic tumor model of melanoma and non-small cell lung cancer lung metastasis, we demonstrate that radiation causes a significant increase in markers of premature senescence in lung parenchyma within 4 to 8 weeks. Further, injection of B16F0 (melanoma) or Lewis Lung carcinoma (epidermoid lung cancer) cells at these time points after radiation results in an increase in the number and size of pulmonary tumor nodules relative to unirradiated mice. Treatment of irradiated mice with a senolytic agent (ABT-737) or agents that prevent senescence (rapamycin, INK-128) was sufficient to reduce radiation-induced lung parenchymal senescence and to mitigate radiation-enhanced tumor growth. These agents abrogated radiation-induced expression of 12-Lipoxygenase (12-LOX), a molecule implicated in several deleterious effects of senescence. Deficiency of 12-LOX prevented radiation-enhanced tumor growth. Together, these data demonstrate the pro-tumorigenic role of radiation-induced senescence, introduces the dual TORC inhibitor INK-128 as an effective agent for prevention of radiation-induced normal tissue senescence, and identifies senescence-associated 12-LOX activity as an important component of the pro-tumorigenic irradiated tissue microenvironment. These studies suggest that combining senotherapeutic agents with radiotherapy may decrease post-therapy tumor growth.
Subject(s)
Carcinoma, Lewis Lung , Lung Neoplasms , Melanoma, Experimental , Animals , Arachidonate 12-Lipoxygenase/pharmacology , Carcinoma, Lewis Lung/enzymology , Carcinoma, Lewis Lung/pathology , Cell Growth Processes , Cellular Senescence , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Melanoma, Experimental/enzymology , Melanoma, Experimental/pathology , Mice , Tumor MicroenvironmentABSTRACT
Glioblastoma is the the most common primary brain tumor in adults. Onset of disease is followed by a uniformly lethal prognosis and dismal overall survival. While immunotherapies have revolutionized treatment in other difficult-to-treat cancers, these have failed to demonstrate significant clinical benefit in patients with glioblastoma. Obstacles to success include the heterogeneous tumor microenvironment (TME), the immune-privileged intracranial space, the blood-brain barrier (BBB) and local and systemic immunosuppressions. Monoclonal antibody-based therapies have failed at least in part due to their inability to access the intracranial compartment. Bispecific T-cell engagers are promising antibody fragment-based therapies which can bring T cells close to their target and capture them with a high binding affinity. They can redirect the entire repertoire of T cells against tumor, independent of T-cell receptor specificity. However, the multiple challenges posed by the TME, immune privilege and the BBB suggest that a single agent approach may be insufficient to yield durable, long-lasting antitumor efficacy. In this review, we discuss the mechanism of action of T-cell engagers, their preclinical and clinical developments to date. We also draw comparisons with other classes of multispecific antibodies and potential combinations using these antibody fragment therapies.
Subject(s)
Antibodies, Bispecific/therapeutic use , Brain Neoplasms/genetics , Glioblastoma/genetics , T-Lymphocytes/immunology , Antibodies, Bispecific/pharmacology , HumansABSTRACT
We have only recently begun to understand how cancer metabolism affects antitumor responses and immunotherapy outcomes. Certain immunometabolic targets have been actively pursued in other tumor types, however, glioblastoma research has been slow to exploit the therapeutic vulnerabilities of immunometabolism. In this review, we highlight the pathways that are most relevant to glioblastoma and focus on how these immunometabolic pathways influence tumor growth and immune suppression. We discuss hypoxia, glycolysis, tryptophan metabolism, arginine metabolism, 2-Hydroxyglutarate (2HG) metabolism, adenosine metabolism, and altered phospholipid metabolism, in order to provide an analysis and overview of the field of glioblastoma immunometabolism.
ABSTRACT
PURPOSE: Type II pneumocyte (alveolar epithelial cells type II [AECII]) senescence has been implicated in the progression of lung fibrosis. The capacity of senescent cells to modulate pulmonary macrophages to drive fibrosis is unexplored. Insulin-like growth factor-1 receptor (IGF-1R) signaling has been implicated as a regulator of senescence and aging. METHODS AND MATERIALS: Mice with an AECII-specific deletion of IGF-1R received thoracic irradiation (n ≥ 5 per condition), and the effect of IGF-1R deficiency on radiation-induced AECII senescence and macrophage polarization to an alternatively activated phenotype (M2) was investigated. IGF-1R signaling, macrophage polarization, and senescence were evaluated in surgically resected human lung (n = 63). RESULTS: IGF-1R deficient mice demonstrated reduced AECII senescence (senescent AECII/field; intact: 7.25% ± 3.5% [mean ± SD], deficient: 2.75% ± 2.8%, P = .0001), reduced accumulation of M2 macrophages (intact: 24.7 ± 2.2 cells/field, deficient: 15.5 ± 1.2 cells/field, P = .0086), and fibrosis (hydroxyproline content; intact: 71.9 ± 21.7 µg/lung, deficient: 31.7 ± 7.9, P = .0485) after irradiation. Senescent AECII enhanced M2 polarization in a paracrine fashion (relative Arg1 mRNA, 0 Gy: 1.0 ± 0.4, 17.5 Gy: 7.34 ± 0.5, P < .0001). Evaluation of surgical samples from patients treated with chemoradiation demonstrated increased expression of IGF-1 (unirradiated: 10.2% ± 4.9% area, irradiated: 15.1% ± 11.5%, P = .0377), p21 (unirradiated: 0.013 ± 0.02 histoscore, irradiated: 0.084 ± 0.09 histoscore, P = .0002), IL-13 (unirradiated: 13.7% ± 2.8% area, irradiated: 21.7% ± 3.8%, P < .0001), and M2 macrophages in fibrotic regions relative to nonfibrotic regions (unirradiated: 11.4 ± 12.2 CD163 + cells/core, irradiated: 43.1 ± 40.9 cells/core, P = .0011), consistent with findings from animal models of lung fibrosis. CONCLUSIONS: This study demonstrates that senescent AECII are necessary for the progression of pulmonary fibrosis and serve as a targetable, chronic stimuli for macrophage activation in fibrotic lung.
Subject(s)
Alveolar Epithelial Cells/physiology , Cell Polarity , Cellular Senescence/physiology , Macrophages, Alveolar/physiology , Pulmonary Fibrosis/etiology , Receptor, IGF Type 1/metabolism , Alveolar Epithelial Cells/radiation effects , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/therapy , Cellular Senescence/radiation effects , Chemoradiotherapy , Gene Deletion , Humans , Hydroxyproline/analysis , Lung/metabolism , Lung/radiation effects , Lung Neoplasms/pathology , Lung Neoplasms/therapy , Macrophage Activation , Macrophages, Alveolar/radiation effects , Mice , Mice, Inbred C57BL , Pulmonary Fibrosis/pathology , Radiation Injuries, Experimental/metabolism , Radiation Injuries, Experimental/physiopathology , Radiation Injuries, Experimental/prevention & control , Receptor, IGF Type 1/deficiency , Receptor, IGF Type 1/geneticsABSTRACT
This study used a nitroaliphatic chemistry approach to synthesize a novel artemisinin-derived carba-dimer (AG-1) and determined its anti-proliferative effects in human normal and cancer cells. AG-1 treatments selectively inhibit proliferation of cancer cells compared to normal human fibroblasts. Compared to artemisinin, AG-1 is more toxic to human breast, prostate, head-neck, pancreas and skin cancer cells; 50% inhibition (IC50) 123 µM in AG-1 vs. 290 µM in artemisinin-treated breast cancer cells. AG-1 treatment decreased (~ 5 folds) cyclin D1 protein expression that correlated with an increase in the percentage of cells in the G1-phase, suggesting a G1 delay. AG-1-induced toxicity was independent of the DNA damage at 72 h post-treatment, as measured by micronuclei frequency and H2AX protein levels. Results from electron paramagnetic resonance spectroscopy showed Fe-catalyzed formation of AG-1 carbon-centered radicals in a cell-free system. Flow cytometry analysis of H2DCF-DA oxidation showed a significant increase in the steady-state levels of reactive oxygen species (ROS) in AG-1-treated cells. Pre-treatment with N-acetyl-l-cysteine and antioxidant enzymes (superoxide dismutase and catalase) significantly suppressed AG-1-induced toxicity, suggesting that superoxide and hydrogen peroxide contribute to AG-1-induced toxicity in human cancer cells. AG-1 represents a novel class of anti-cancer drug that is more potent than its parent compound, artemisinin.
ABSTRACT
Radiation-induced pulmonary fibrosis (RIPF) is a chronic, progressive complication of therapeutic irradiation of the thorax. It has been suggested that senescence of type II pneumocytes (AECIIs), an alveolar stem cell, plays a role in the development of RIPF through loss of replicative reserve and via senescent AECII-driven release of proinflammatory and profibrotic cytokines. Within this context, we hypothesized that arachidonate 12-lipoxygenase (12-LOX) is a critical mediator of AECII senescence and RIPF. Treatment of wild-type AECIIs with 12S-hydroxyeicosateraenoic acid (12S-HETE), a downstream product of 12-LOX, was sufficient to induce senescence in a NADPH oxidase 4 (NOX4)-dependent manner. Mice deficient in 12-LOX exhibited reduced AECII senescence, pulmonary collagen accumulation and accumulation of alternatively activated (M2) macrophages after thoracic irradiation (5 × 6 Gy) compared to wild-type mice. Conditioned media from irradiated or 12S-HETE-treated primary pneumocytes contained elevated levels of IL-4 and IL-13 compared to untreated pneumocytes. Primary macrophages treated with conditioned media from irradiated AECII demonstrated preferential M2 type polarization when AECIIs were derived from wild-type mice compared to 12-LOX-deficient mice. Together, these data identified 12-LOX as a critical component of RIPF and a therapeutic target for radiation-induced lung injury.
Subject(s)
Alveolar Epithelial Cells/pathology , Arachidonate 12-Lipoxygenase/metabolism , Cellular Senescence/radiation effects , Macrophages/radiation effects , Radiation Pneumonitis/enzymology , Alveolar Epithelial Cells/radiation effects , Animals , Arachidonate 12-Lipoxygenase/genetics , Female , Gene Expression Regulation, Enzymologic/radiation effects , Interleukin-13/biosynthesis , Interleukin-4/biosynthesis , Macrophages/cytology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Radiation Pneumonitis/genetics , Radiation Pneumonitis/immunology , Radiation Pneumonitis/pathologyABSTRACT
Adaptation of tumor cells to radiotherapy induces changes that are actionable by molecular targeted agents and immunotherapy. This report demonstrates that radiation-induced changes in integrin expression can be targeted 2 months later. Integrins are transmembrane cell adhesion molecules that are essential for cancer cell survival and proliferation. To analyze the short- and long-term effects of radiation on the integrin expression, prostate cancer cells (DU145, PC3, and LNCaP) were cultured in a 3D extracellular matrix and irradiated with either a single dose of radiation (2-10 Gy) or a multifractionated regimen (2-10 fractions of 1 Gy). Whole human genome microarrays, immunoblotting, immunoprecipitation assays, and immunofluorescence staining of integrins were performed. The results were confirmed in a prostate cancer xenograft model system. Interestingly, ß1 and ß4 integrins (ITGB1 and ITGB4) were upregulated after radiation in vitro and in vivo. This overexpression lasted for more than 2 months and was dose dependent. Moreover, radiation-induced upregulation of ß1 and ß4 integrin resulted in significantly increased tumor cell death after treatment with inhibitory antibodies. Combined, these findings indicate that long-term tumor adaptation to radiation can result in an increased susceptibility of surviving cancer cells to molecular targeted therapy due to a radiation-induced overexpression of the target. IMPLICATIONS: Radiation induces dose- and schedule-dependent adaptive changes that are targetable for an extended time; thus suggesting radiotherapy as a unique strategy to orchestrate molecular processes, thereby providing new radiation-drug treatment options within precision cancer medicine.
Subject(s)
Antineoplastic Agents, Immunological/administration & dosage , Integrin beta Chains/genetics , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/radiotherapy , Animals , Antineoplastic Agents, Immunological/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Cell Survival/drug effects , Cell Survival/radiation effects , Combined Modality Therapy , Dose-Response Relationship, Radiation , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Integrin beta Chains/metabolism , Integrin beta1/genetics , Integrin beta1/metabolism , Integrin beta4/genetics , Integrin beta4/metabolism , Male , Mice , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Radiation Tolerance/drug effects , Up-Regulation/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Therapeutic advances have markedly prolonged overall survival in multiple myeloma (MM) but the disease currently remains incurable. In a panel of MM cell lines (MM.1S, OPM-2, H929, and U266), using CD138 immunophenotyping, side population staining, and stem cell-related gene expression, we demonstrate the presence of stem-like tumor cells. Hypoxic culture conditions further increased CD138low stem-like cells with upregulated expression of OCT4 and NANOG. Compared to MM cells, these stem-like cells maintained lower steady-state pro-oxidant levels with increased uptake of the fluorescent deoxyglucose analog. In primary human MM samples, increased glycolytic gene expression correlated with poorer overall and event-free survival outcomes. Notably, stem-like cells showed increased mitochondrial mass, rhodamine 123 accumulation, and orthodox mitochondrial configuration while more condensed mitochondria were noted in the CD138high cells. Glycolytic inhibitor 2-deoxyglucose (2-DG) induced ER stress as detected by qPCR (BiP, ATF4) and immunoblotting (BiP, CHOP) and increased dihydroethidium probe oxidation both CD138low and CD138high cells. Treatment with a mitochondrial-targeting agent decyl-triphenylphosphonium (10-TPP) increased intracellular steady-state pro-oxidant levels in stem-like and mature MM cells. Furthermore, 10-TPP mediated increases in mitochondrial oxidant production were suppressed by ectopic expression of manganese superoxide dismutase. Relative to 2-DG or 10-TPP alone, 2-DG plus 10-TPP combination showed increased caspase 3 activation in MM cells with minimal toxicity to the normal hematopoietic progenitor cells. Notably, treatment with polyethylene glycol conjugated catalase significantly reduced 2-DG and/or 10-TPP-induced apoptosis of MM cells. Also, the combination of 2-DG with 10-TPP decreased clonogenic survival of MM cells. Taken together, this study provides a novel strategy of metabolic oxidative stress-induced cytotoxicity of MM cells via 2-DG and 10-TPP combination therapy.
Subject(s)
Cell Death/drug effects , Deoxyglucose/pharmacology , Multiple Myeloma/pathology , Neoplastic Stem Cells/pathology , Onium Compounds/pharmacology , Organophosphorus Compounds/pharmacology , Oxidative Stress/drug effects , Biological Transport/drug effects , Cell Hypoxia/drug effects , Cell Line, Tumor , Drug Interactions , Endoplasmic Reticulum Stress/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Glucose/metabolism , Glycolysis/genetics , Humans , Neoplastic Stem Cells/drug effects , Prognosis , Reactive Oxygen Species/metabolismABSTRACT
Metastatic melanoma is the most aggressive and lethal form of skin cancer. Emerging evidence suggests that differences in melanoma metabolism relative to nonmalignant cells represent potential targets for improved therapy for melanoma. Specifically, melanoma cells exhibit increased mitochondrial electron transport chain (ETC) activity and concomitant hyperpolarized mitochondrial membrane potential relative to nonmalignant cells. We have synthesized several new fluorescent lipophilic vinylpyridinium cations built from tetraarylethylene scaffolds that target mitochondria via attraction to the hyperpolarized mitochondrial membrane potential. Mitochondria-specific accumulation in melanoma cells relative to normal human fibroblasts was demonstrated using confocal fluorescence microscopy and resulted in the disruption of oxidative metabolism leading to melanoma specific cell death in vitro. Thus, the pyridinium tetraarylethylene platform represents a promising new mitochondrial-targeted delivery vehicle with potential imaging and therapeutic properties.