Membrane-bound Tumor Necrosis Factor Research Articles

Abstract P1163: Identifying Novel Intracellular Interactions Driving Membrane-bound Tumour Necrosis Factor-mediated Signalling In The Microvasculature

Elevated total peripheral resistance (TPR) is a hallmark of many cardiovascular diseases and furthers disease progression. We previously demonstrated that myogenic tone, regulated by membrane-bound tumour necrosis factor (mTNF) reverse signalling, is a primary modulator of TPR. Thus, inhibiting mTNF reverse signalling should reduce TPR with the potential to improve disease outcome. However, since mTNF is a ubiquitous protein with critical biological functions, its indiscriminate inhibition can cause widespread side effects. Therefore, therapeutics aiming to reduce myogenic tone and TPR must target other elements of mTNF reverse signalling. To identify these unknown participants of mTNF reverse signalling, we used proximity-dependent biotinylation coupled to mass spectrometry (BioID). Our search identified 42 high confidence hits that interact with and/or are proximal to mTNF’s cytoplasmic domain. Gene ontology (GO) analysis revealed that molecularly, these proteins are structural constituents of the cytoskeleton involved in actin and cell adhesion molecule binding. This aligns with our underlying hypothesis that mTNF functions as a mechanosensor that tethers to its receptors and initiates myogenic vasoconstriction in response to changes in transmural pressure. GO also identified proteins involved in MAPK and RhoA signalling, which are critically involved in myogenic vasoconstriction. Our most abundant hit (fold change = 12.6, p-value = 0.0023) is a cytoskeletal protein that plays a role in maintaining the structural integrity of cells. This protein also interacts with proteins involved in various signalling cascades, namely, RhoA and calcium-dependent signalling pathways. Thus, we hypothesize that this protein is essential for formation of signalling complexes with mTNF and interacts with other proteins to mediate reverse signalling . We will employ a bioluminescence-based approach to quantify the strength of all interactions detected by BioID and identify small molecule inhibitors that can disrupt these interactions. These newly discovered inhibitors could potentially be used as novel therapeutics targeting myogenic tone and hence, elevated TPR to improve disease management and clinical outcome of cardiovascular diseases.

Complexity of immunobiology of tumor necrosis factor and novel anti-TNF therapy

Tumor Necrosis Factor (TNF) was discovered almost 50 years ago as “serum factor” detected in mice following infections or administration of bacterial lipopolysaccharide (LPS), with a remarkable anti-tumor effect. Molecular cloning showed that this activity is mediated by a small protein (17 kDa), which belongs to a wide plethora of cytokines. Due to the particular organization of the TNF gene coding sequence, all cells producing soluble TNF also carry a membrane-bound cytokine on their surface. The physiological effects of TNF are mediated by signaling through two types of highly specific receptors. Despite established protective and homeostatic functions of TNF, when overproduced systemically or locally, it can trigger pathologies ranging from septic shock to autoimmune diseases. Therefore, in clinical immunotherapy there were not the TNF agonists, which were expected to induce anti-tumor effects, but rather the antagonistic blockers, that proved effective in a wide range of autoimmune diseases with an inflammatory component. Our studies in mice based on the technologies of reverse genetics and experimental disease models, revealed a paradoxical feature of TNF: some cellular sources of this cytokine (such as myeloid cells) promoted diseases, while other cell types (such as T lymphocytes) produced a protective form of the same cytokine. There are several possible mechanistic explanations for this phenomenon. On the one hand, the "pathogenic" cytokine is produced in a soluble form and can exert systemic effects via broadly expressed TNFR1. On the other hand, protective functions are mediated by the membrane-bound TNF via TNFR2. Systemic anti-cytokine therapy is known to be accompanied by undesirable side effects, which can hypothetically be explained by the neutralization of these protective functions. Thus, we developed prototypes of TNF blockers which limit the bioavailability of this cytokine only from its main “pathogenic” source – myeloid cells. This type of inhibitors, called MYSTI, represent bispecific mini-antibodies binding both TNF and a surface marker on myeloid cells and lacking the Fc domain. MYSTI retain newly synthesized TNF on the surface of the producing cell and then internalize it. This novel type of immunotherapy drug has already shown efficacy in a number of experimental disease models.

Membrane Protein Binding Interactions Studied in Live Cells via Diethylpyrocarbonate Covalent Labeling Mass Spectrometry.

Membrane proteins are vital in the human proteome for their cellular functions and make up a majority of drug targets in the U.S. However, characterizing their higher-order structures and interactions remains challenging. Most often membrane proteins are studied in artificial membranes, but such artificial systems do not fully account for the diversity of components present in cell membranes. In this study, we demonstrate that diethylpyrocarbonate (DEPC) covalent labeling mass spectrometry can provide binding site information for membrane proteins in living cells using membrane-bound tumor necrosis factor α (mTNFα) as a model system. Using three therapeutic monoclonal antibodies that bind TNFα, our results show that residues that are buried in the epitope upon antibody binding generally decrease in DEPC labeling extent. Additionally, serine, threonine, and tyrosine residues on the periphery of the epitope increase in labeling upon antibody binding because of a more hydrophobic microenvironment that is created. We also observe changes in labeling away from the epitope, indicating changes to the packing of the mTNFα homotrimer, compaction of the mTNFα trimer against the cell membrane, and/or previously uncharacterized allosteric changes upon antibody binding. Overall, DEPC-based covalent labeling mass spectrometry offers an effective means of characterizing structure and interactions of membrane proteins in living cells.

Interaction between tumor cell TNFR2 and monocyte membrane-bound TNF-α triggers tumorigenic inflammation in neuroblastoma

BackgroundTumor progression and resistance to therapy in children with neuroblastoma (NB), a common childhood cancer, are often associated with infiltration of monocytes and macrophages that produce inflammatory cytokines. However, the...

TNF hampers intestinal tissue repair in colitis by restricting IL-22 bioavailability

Successful treatment of chronic inflammatory diseases integrates both the cessation of inflammation and the induction of adequate tissue repair processes. Strikingly, targeting a single proinflammatory cytokine, tumor necrosis factor (TNF), induces both processes in a relevant cohort of inflammatory bowel disease (IBD) patients. However, the molecular mechanisms underlying intestinal repair following TNF blockade during IBD remain elusive. Using a novel humanized model of experimental colitis, we demonstrate that TNF interfered with the tissue repair program via induction of a soluble natural antagonist of IL-22 (IL-22Ra2; IL-22BP) in the colon and abrogated IL-22/STAT3-mediated mucosal repair during colitis. Furthermore, membrane-bound TNF expressed by T cells perpetuated colonic inflammation, while soluble TNF produced by epithelial cells (IECs) induced IL-22BP expression in colonic dendritic cells (DCs) and dampened IL-22-driven restitution of colonic epithelial functions. Finally, TNF induced IL-22BP expression in human monocyte-derived DCs and levels of IL22-BP correlated with TNF in sera of IBD patients. Thus, our data can explain how anti-TNF therapy induces mucosal healing by increasing IL-22 availability and implicates new therapeutic opportunities for IBD.

TNFR2 pathways are fully active in cancer regulatory T cells.

Tumor necrosis factor receptor 2 (TNFR2), a membrane-bound tumor necrosis factor receptor expressed by regulatory T cells (Tregs), participates in Treg proliferation. Although a specific TNFR2 pathway has been reported, the signaling mechanism has not been completely elucidated. This study sought to clarify TNFR2 signaling in human Tregs using amplicon sequencing and single-cell RNA sequencing to assess Tregs treated with a TNFR2 agonist antibody. Pathway enrichment analysis based on differentially expressed genes highlighted tumor necrosis factor α signaling via nuclear factor kappa B, interleukin-2 signal transducer and activator of transcription 5 signaling, interferon-γ response, and cell proliferation-related pathways in Tregs after TNFR2 activation. TNFR2-high Treg-focused analysis found that these pathways were fully activated in cancer Tregs, showing high TNFR2 expression. Collectively, these findings suggest that TNFR2 orchestrates multiple pathways in cancer Tregs, which could help cancer cells escape immune surveillance, making TNFR2 signaling a potential anticancer therapy target.

The role of soluble TNF in mediating immune and metabolic alterations in a mouse model of amyloid-beta pathology.

Insulin resistance (IR) and systemic inflammation are risk factors for Alzheimer's disease (AD); however, the molecular and signaling mechanisms underlying this relationship are not well understood. Elevated plasma cytokine levels are associated with increased risk for both AD and IR. The cytokine tumor necrosis factor (TNF) is a major driver of inflammation and is implicated in IR and neurodegeneration. Here, we hypothesize that soluble TNF (solTNF) drives metabolic and immune alterations in the CNS and periphery that promote IR, thereby increasing the risk for AD pathologies. To investigate this hypothesis, 2-month old female 5xFAD mice were fed a high-fat high-carbohydrate diet (HFHC) or a control diet (CD) for 8 weeks. After 1 month, the brain-permeant solTNF inhibitor XPro1595 or a brain-impermeant pan (sol and membrane-bound) TNF inhibitor Etanercept or saline were dosed twice weekly for 4 weeks. Plasma, adipose tissue, and gut were collected for evaluation of IR, metabolic and immune parameters. Molecular markers of insulin signaling, oxidative stress, mitochondrial impairment and inflammation were assessed in relevant brain regions (hippocampus, hypothalamus and cortex). Our preliminary data demonstrate that HFHC-fed 5XFAD mice exhibit increased body weight gain compared to CD groups. Ectopic lipid deposition, as measured by gonadal fat pad and liver volumes, was significantly increased, concurrent to anatomical indicators of intestinal inflammation, including shortened small intestine and colon. HFHC diet promoted systemic inflammation, as measured by increases in the classical pro-inflammatory cytokines IL-6 and TNF. Further, plasma LCN2, a downstream TNF inflammatory molecule associated with insulin insensitivity, hippocampal alterations, and previously observed to be increased in AD patients, was also significantly increased. Ongoing assessments of mRNA and protein expression of insulin signaling and inflammation in the hippocampus and cortex will determine additional effects of TNF in central insulin impairment and AD pathogenesis and will be presented and integrated into the overall findings. Together, these data suggest HFHC diet disrupts peripheral immune and metabolic parameters in the 5XFAD animal model of AD-like pathology, thereby triggering neuroinflammation and insulin insensitivity, conditions which have been implicated in significantly increasing the risk for development of AD in humans.

Experimental Subarachnoid Hemorrhage Drives Catecholamine-Dependent Cardiac and Peripheral Microvascular Dysfunction.

Subarachnoid hemorrhage (SAH) is a devastating cerebral event caused by an aneurysmal rupture. In addition to neurological injury, SAH has significant effects on cardiac function and the peripheral microcirculation. Since these peripheral complications may exacerbate brain injury, the prevention and management of these peripheral effects are important for improving the overall clinical outcome after SAH. In this investigation, we examined the effects of SAH on cardiac function and vascular reactivity in a well-characterized blood injection model of SAH. Standard echocardiographic and blood pressure measurement procedures were utilized to assess cardiac function and hemodynamic parameters in vivo; we utilized a pressure myography approach to assess vascular reactivity in cremaster skeletal muscle resistance arteries ex vivo. We observed that elevated catecholamine levels in SAH stun the myocardium, reduce cardiac output and augment myogenic vasoconstriction in isolated cremaster arteries. These cardiac and vascular effects are driven by beta- and alpha-adrenergic receptor signaling, respectively. Clinically utilized adrenergic receptor antagonists can prevent cardiac injury and normalize vascular function. We found that tumor necrosis factor (TNF) gene deletion prevents the augmentation of myogenic reactivity in SAH: since membrane-bound TNF serves as a mechanosensor in the arteries assessed, alpha-adrenergic signaling putatively augments myogenic vasoconstriction by enhancing mechanosensor activity.

In vitro and in vivo discrepancy in inducing apoptosis by mesenchymal stromal cells delivering membrane-bound tumor necrosis factor–related apoptosis inducing ligand in osteosarcoma pre-clinical models

BackgroundOsteosarcoma (OS) is the most frequent pediatric malignant bone tumor. OS patients have not seen any major therapeutic progress in the last 30 years, in particular in the case of metastatic disease, which requires new therapeutic strategies. The pro-apoptotic cytokine Tumor necrosis factor (TNF)–Related Apoptosis Inducing Ligand (TRAIL) can selectively kill tumor cells while sparing normal cells, making it a promising therapeutic tool in several types of cancer. However, many OS cell lines appear resistant to recombinant human (rh)TRAIL-induced apoptosis. We, therefore, hypothesized that TRAIL presentation at the membrane level of carrier cells might overcome this resistance and trigger apoptosis. MethodsTo address this, human adipose mesenchymal stromal cells (MSCs) transfected in a stable manner to express membrane-bound full-length human TRAIL (mbTRAIL) were co-cultured with several human OS cell lines. ResultsThis induced apoptosis by cell-to-cell contact even in cell lines initially resistant to rhTRAIL. In contrast, mbTRAIL delivered by MSCs was not able to counteract tumor progression in this OS orthotopic model. DiscussionThis was partly due to the fact that MSCs showed a potential to support tumor development. Moreover, the expression of mbTRAIL did not show caspase activation in adjacent tumor cells.

Critical Role of Tumor Necrosis Factor Signaling in Mesenchymal Stem Cell-Based Therapy for Autoimmune and Inflammatory Diseases.

Mesenchymal stem cells (MSCs) have been broadly used as a therapy for autoimmune disease in both animal models and clinical trials. MSCs inhibit T effector cells and many other immune cells, while activating regulatory T cells, thus reducing the production of pro-inflammatory cytokines, including tumor necrosis factor (TNF), and repressing inflammation. TNF can modify the MSC effects via two TNF receptors, i.e., TNFR1 in general mediates pro-inflammatory effects and TNFR2 mediates anti-inflammatory effects. In the central nervous system, TNF signaling plays a dual role, which enhances inflammation via TNFR1 on immune cells while providing cytoprotection via TNFR2 on neural cells. In addition, the soluble form of TNFR1 and membrane-bound TNF also participate in the regulation to fine-tune the functions of target cells. Other factors that impact TNF signaling and MSC functions include the gender of the host, disease course, cytokine concentrations, and the length of treatment time. This review will introduce the fascinating progress in this aspect of research and discuss remaining questions and future perspectives.

Constitutive smooth muscle tumour necrosis factor regulates microvascular myogenic responsiveness and systemic blood pressure

Tumour necrosis factor (TNF) is a ubiquitously expressed cytokine with functions beyond the immune system. In several diseases, the induction of TNF expression in resistance artery smooth muscle cells enhances microvascular myogenic vasoconstriction and perturbs blood flow. This pathological role prompted our hypothesis that constitutively expressed TNF regulates myogenic signalling and systemic haemodynamics under non-pathological settings. Here we show that acutely deleting the TNF gene in smooth muscle cells or pharmacologically scavenging TNF with etanercept (ETN) reduces blood pressure and resistance artery myogenic responsiveness; the latter effect is conserved across five species, including humans. Changes in transmural pressure are transduced into intracellular signals by membrane-bound TNF (mTNF) that connect to a canonical myogenic signalling pathway. Our data positions mTNF ‘reverse signalling’ as an integral element of a microvascular mechanosensor; pathologic or therapeutic perturbations of TNF signalling, therefore, necessarily affect microvascular tone and systemic haemodynamics.

Tumor Necrosis Factor: Function, Release and Clearance.

Tumor Necrosis Factor (TNF) is a multifunctional cytokine. It plays an important role in the pathophysiology of several diseases. Recently, it has been discovered that TNF is circulating in two different forms, a bioactive form and an immunologically detectable form. These two forms of TNF show different clearance kinetics. The immunological form is supposed to be an inactivated TNF protein. For this inactivation, proteolytic degradation or TNF binding by inactivating proteins is necessary. In this review we have focused on TNF inactivation by TNF binding proteins. Recent data show that there are soluble TNF receptors circulating which can bind and inactivate TNF. These receptors are membrane-bound TNF receptors which have been proteolytically cleaved from the cell membrane. Two TNF receptors are circulating, the soluble TNF receptor of 55 kDa (P55) and the receptor of 75 kDa (P75). The receptors are held responsible not only for inactivation of the TNF, but also for the clearance of TNF. Recent data show that the kidney is the most important organ for TNF clearance, followed by the liver. All other organs are of less importance. In this review, function, release, and clearance of TNF are discussed.

Comparable Immune Function Inhibition by the Infliximab Biosimilar CT-P13: Implications for Treatment of Inflammatory Bowel Disease.

CT-P13 is the first biosimilar monoclonal antibody to infliximab, and was recently approved in the European Union, Japan, Korea, and USA for all six indications of infliximab. However, studies directly assessing the biologic activity of CT-P13 versus inflximab in the context of inflammatory bowel disease [IBD] are still scanty. In the present study, we aimed to compare the biological activities of CT-P13 and infliximab with specific focus on intestinal cells so as to gain insight into the potential biosimilarity of these two agents for treatment of IBD. CT-P13 and infliximab were investigated and compared by in vitro experiments for their neutralisation ability of soluble tumour necrosis factor alpha [sTNFα] and membrane-bound tumour necrosis factor alpha [mTNFα], suppression of cytokine release by reverse signalling, induction of regulatory macrophages and wound healing, and antibody-dependent cell cytotoxicity [ADCC]. CT-P13 showed similar biological activities to infliximab as gauged by neutralisation of soluble TNFα, as well as blockade of apoptosis and suppression of pro-inflammatory cytokines in intestinal Caco-2 cells. Infliximab and CT-P13 equally induced apoptosis and outside-to-inside signals through transmembrane TNFα [tmTNFα]. Moreover, regulatory macrophage induction and ensuing wound healing were similarly exerted by CT-P13 and infliximab. However, neither CT-P13 nor infliximab exerted any significant ADCC of ex vivo-stimulated peripheral blood monocytes or lamina propria mononuclear cells from IBD patients. These findings indicate that CT-P13 and infliximab exert highly similar biological activities in intestinal cells, and further support a mechanistic comparability of these two drugs in the treatment of IBD.

Confocal Laser Endomicroscopy for Membrane-Bound Tumor Necrosis Factor Predicts Response to Therapy in Crohn's Disease

Confocal Laser Endomicroscopy for Membrane-Bound Tumor Necrosis Factor Predicts Response to Therapy in Crohn's Disease

Fluorescent antibodies to membrane-bound tumor necrosis factor (mTNF) as a predictive biomarker for therapeutic responders

Fluorescent antibodies to membrane-bound tumor necrosis factor (mTNF) as a predictive biomarker for therapeutic responders

P2X7 Receptor and Cytokines Contribute to Extra-territorial Facial Pain

The whisker pad area (WP) is innervated by the second branch of the trigeminal nerve and experiences allodynia and hyperalgesia following transection of the mental nerve (MN; the third branch of the trigeminal nerve). However, the mechanisms of this extra-territorial pain remain unclear. The ionotropic P2X7 ATP receptor (P2X7) in microglia is known to potentiate, via cytokines, the perception of noxious stimuli, raising the possibility that P2X7 and cytokines are involved in this extra-territorial pain. One day after MN transection (MNT), WP allodynia/hyperalgesia developed, which lasted for > 8 wks. Activation of microglia and up-regulation of P2X7, membrane-bound tumor necrosis factor (TNF)-α (mTNF-α), and soluble TNF-α (sTNF-α) in the trigeminal sensory nuclear complex (TNC) were evident for up to 6 wks after MNT. Allodynia/hyperalgesia after MNT was blocked by intracisternal administration of etanercept, a recombinant TNF-α receptor (p75)-Fc fusion protein. Intracisternal A438079, a P2X7 antagonist, also attenuated allodynia/hyperalgesia and blocked up-regulation of mTNF-α and sTNF-α in the TNC. We conclude that sTNF-α released by microglia following P2X7 activation may be important in both the initiation and maintenance of extra-territorial pain after MNT.

Antibodies Against Tumor Necrosis Factor (TNF) Induce T-Cell Apoptosis in Patients With Inflammatory Bowel Diseases via TNF Receptor 2 and Intestinal CD14+ Macrophages

The anti-tumor necrosis factor (TNF) antibodies infliximab, adalimumab, and certolizumab pegol have proven clinical efficacy in Crohn's disease. Here, we assessed the effects of anti-TNF antibodies on apoptosis in inflammatory bowel disease (IBD). CD14(+) macrophages and CD4(+) T cells were isolated from peripheral blood and lamina propria mononuclear cells from patients with IBD and control patients. Cell surface markers and apoptosis were assessed by immunohistology and fluorescence-activated cell sorting techniques. Lamina propria CD14(+) macrophages showed significantly more frequent and higher membrane-bound TNF (mTNF) expression than CD4(+) T cells in IBD, whereas mTNF-dependent signaling proteins such as TNF receptor (TNFR) 2, TNFR-associated factor (TRAF) 2, and nuclear factor κB were induced in IBD mucosal CD4(+) T cells. Most anti-TNF antibodies did not induce T-cell apoptosis in purified peripheral or mucosal CD4(+) T cells. However, in contrast to etanercept, administration of all clinically effective anti-TNF antibodies resulted in a significant induction of T-cell apoptosis in IBD when lamina propria CD4(+) T cells expressing TNFR2(+) were cocultured with mTNF(+) CD14(+) intestinal macrophages. In contrast, no effects in control patients were noted. T-cell apoptosis in IBD occurred in vivo after treatment with adalimumab and infliximab, was critically dependent on TNFR2 signaling, and could be prevented via interleukin-6 signal transduction. Blockade of interleukin-6R signaling augmented anti-TNF-induced T-cell apoptosis in IBD. Clinically effective anti-TNF antibodies are able to induce T-cell apoptosis in IBD only when mucosal TNFR2(+) T cells are cocultured with mTNF-expressing CD14(+) macrophages. The finding that anti-TNF antibodies induce apoptosis indirectly by targeting the mTNF/TNFR2 pathway may have important implications for the development of new therapeutic strategies in IBD.

Isolation of viable antigen-specific CD8 + T cells based on membrane-bound tumor necrosis factor (TNF)-α expression

Current technology to isolate viable cytokine-producing antigen-specific primary human T cells is limited to bi-specific antibody capture systems, which suffer from limited sensitivity and high background. Here, we describe a novel procedure for isolating antigen-specific human T cells based on their ability to produce tumor necrosis factor (TNF)-α. Unlike many cytokines, TNF-α is initially produced in a biologically active membrane-bound form that is subsequently cleaved by TNF-α converting enzyme (TACE) to release the soluble form of TNF-α. By preventing this cleavage event, we show that TNF-α can be ‘trapped’ on the surface of the T cells from which it originates and directly labeled for viable isolation of these antigen-specific T cells. Together with other existing sorting procedures to isolate activated T cells, this new technique should permit the direct isolation of multi-functional T lymphocytes for further protein and gene expression analyses, as well as a detailed functional assessment of the potential role that TNF-α producing T cells play in the adaptive immune system.

Cellular source and molecular form of TNF specify its distinct functions in organization of secondary lymphoid organs

AbstractSecondary lymphoid organs provide a unique microenvironment for generation of immune responses. Using a cell type–specific conditional knockout approach, we have dissected contributions of tumor necrosis factor (TNF) produced by B cells (B-TNF) or T cells (T-TNF) to the genesis and homeostatic organization of secondary lymphoid organs. In spleen, lymph nodes and Peyer patches, the cellular source of TNF, and its molecular form (soluble versus membrane-bound) appeared distinct. In spleen, in addition to major B-TNF signal, a complementary T-TNF signal contributed to the microstructure. In contrast, B-TNF predominantly controlled the development of follicular dendritic cells and B-cell follicles in Peyer patches. In lymph nodes, cooperation between TNF expressed by B and T cells was necessary for the maintenance of microarchitecture and for generation of an efficient humoral immune response. Unexpectedly, soluble but not membrane TNF expressed by B cells was essential for the organization of the secondary lymphoid organs. Thus, the maintenance of each type of secondary lymphoid organ is orchestrated by distinct contributions of membrane-bound and soluble TNF produced by B and T lymphocytes.

Human CD34+ cells engineered to express membrane-bound tumor necrosis factor–related apoptosis-inducing ligand target both tumor cells and tumor vasculature

AbstractAdenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor–related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 105 tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell–derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling–stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.