Tumor detection and elimination by a targeted gallium corrole

Sulfonated gallium(III) corroles are intensely fluorescent macrocyclic compounds that spontaneously assemble with carrier proteins to undergo cell entry. We report in vivo imaging and therapeutic efficacy of a tumor-targeted corrole noncovalently assembled with a heregulin-modified protein directed at the human epidermal growth factor receptor (HER). Systemic delivery of this protein-corrole complex results in tumor accumulation, which can be visualized in vivo owing to intensely red corrole fluorescence. Targeted delivery in vivo leads to tumor cell death while normal tissue is spared. These findings contrast with the effects of doxorubicin, which can elicit cardiac damage during therapy and required direct intratumoral injection to yield similar levels of tumor shrinkage compared with the systemically delivered corrole. The targeted complex ablated tumors at >5 times a lower dose than untargeted systemic doxorubicin, and the corrole did not damage heart tissue. Complexes remained intact in serum and the carrier protein elicited no detectable immunogenicity. The sulfonated gallium(III) corrole functions both for tumor detection and intervention with safety and targeting advantages over standard chemotherapeutic agents

Activated Bone Marrow-Derived Macrophages Eradicate Alzheimer's-Related Aβ42 Oligomers and Protect Synapses.

Impaired synaptic integrity and function due to accumulation of amyloid β-protein (Aβ42) oligomers is thought to be a major contributor to cognitive decline in Alzheimer's disease (AD). However, the exact role of Aβ42 oligomers in synaptotoxicity and the ability of peripheral innate immune cells to rescue synapses remain poorly understood due to the metastable nature of oligomers. Here, we utilized photo-induced cross-linking to stabilize pure oligomers and study their effects vs. fibrils on synapses and protection by Aβ-phagocytic macrophages. We found that cortical neurons were more susceptible to Aβ42 oligomers than fibrils, triggering additional neuritic arborization retraction, functional alterations (hyperactivity and spike waveform), and loss of VGluT1- and PSD95-excitatory synapses. Co-culturing neurons with bone marrow-derived macrophages protected synapses against Aβ42 fibrils; moreover, immune activation with glatiramer acetate (GA) conferred further protection against oligomers. Mechanisms involved increased Aβ42 removal by macrophages, amplified by GA stimulation: fibrils were largely cleared through intracellular CD36/EEA1+-early endosomal proteolysis, while oligomers were primarily removed via extracellular/MMP-9 enzymatic degradation. In vivo studies in GA-immunized or CD115+-monocyte-grafted APPSWE/PS1ΔE9-transgenic mice followed by pre- and postsynaptic analyses of entorhinal cortex and hippocampal substructures corroborated our in vitro findings of macrophage-mediated synaptic preservation. Together, our data demonstrate that activated macrophages effectively clear Aβ42 oligomers and rescue VGluT1/PSD95 synapses, providing rationale for harnessing macrophages to treat AD

HER3-targeted protein chimera forms endosomolytic capsomeres and self-assembles into stealth nucleocapsids for systemic tumor homing of RNA interference in vivo.

RNA interference represents a potent intervention for cancer treatment but requires a robust delivery agent for transporting gene-modulating molecules, such as small interfering RNAs (siRNAs). Although numerous molecular approaches for siRNA delivery are adequate in vitro, delivery to therapeutic targets in vivo is limited by payload integrity, cell targeting, efficient cell uptake, and membrane penetration. We constructed nonviral biomaterials to transport small nucleic acids to cell targets, including tumor cells, on the basis of the self-assembling and cell-penetrating activities of the adenovirus capsid penton base. Our recombinant penton base chimera contains polypeptide domains designed for noncovalent assembly with anionic molecules and tumor homing. Here, structural modeling, molecular dynamics simulations, and functional assays suggest that it forms pentameric units resembling viral capsomeres that assemble into larger capsid-like structures when combined with siRNA cargo. Pentamerization forms a barrel lined with charged residues mediating pH-responsive dissociation and exposing masked domains, providing insight on the endosomolytic mechanism. The therapeutic impact was examined on tumors expressing high levels of HER3/ErbB3 that are resistant to clinical inhibitors. Our findings suggest that our construct may utilize ligand mimicry to avoid host attack and target the siRNA to HER3+ tumors by forming multivalent capsid-like structures

Activated Bone Marrow-Derived Macrophages Eradicate Alzheimer's-Related Aβ₄₂ Oligomers and Protect Synapses

Impaired synaptic integrity and function due to accumulation of amyloid β-protein (Aβ₄₂) oligomers is thought to be a major contributor to cognitive decline in Alzheimer's disease (AD). However, the exact role of Aβ₄₂ oligomers in synaptotoxicity and the ability of peripheral innate immune cells to rescue synapses remain poorly understood due to the metastable nature of oligomers. Here, we utilized photo-induced cross-linking to stabilize pure oligomers and study their effects vs. fibrils on synapses and protection by Aβ-phagocytic macrophages. We found that cortical neurons were more susceptible to Aβ₄₂ oligomers than fibrils, triggering additional neuritic arborization retraction, functional alterations (hyperactivity and spike waveform), and loss of VGluT1- and PSD95-excitatory synapses. Co-culturing neurons with bone marrow-derived macrophages protected synapses against Aβ₄₂ fibrils; moreover, immune activation with glatiramer acetate (GA) conferred further protection against oligomers. Mechanisms involved increased Aβ₄₂ removal by macrophages, amplified by GA stimulation: fibrils were largely cleared through intracellular CD36/EEA1⁺-early endosomal proteolysis, while oligomers were primarily removed via extracellular/MMP-9 enzymatic degradation. In vivo studies in GA-immunized or CD115⁺-monocyte-grafted APP_(SWE)/PS1_(ΔE9)-transgenic mice followed by pre- and postsynaptic analyses of entorhinal cortex and hippocampal substructures corroborated our in vitro findings of macrophage-mediated synaptic preservation. Together, our data demonstrate that activated macrophages effectively clear Aβ₄₂ oligomers and rescue VGluT1/PSD95 synapses, providing rationale for harnessing macrophages to treat AD

Activated Bone Marrow-Derived Macrophages Eradicate Alzheimer's-Related Aβ₄₂ Oligomers and Protect Synapses

Impaired synaptic integrity and function due to accumulation of amyloid β-protein (Aβ₄₂) oligomers is thought to be a major contributor to cognitive decline in Alzheimer's disease (AD). However, the exact role of Aβ₄₂ oligomers in synaptotoxicity and the ability of peripheral innate immune cells to rescue synapses remain poorly understood due to the metastable nature of oligomers. Here, we utilized photo-induced cross-linking to stabilize pure oligomers and study their effects vs. fibrils on synapses and protection by Aβ-phagocytic macrophages. We found that cortical neurons were more susceptible to Aβ₄₂ oligomers than fibrils, triggering additional neuritic arborization retraction, functional alterations (hyperactivity and spike waveform), and loss of VGluT1- and PSD95-excitatory synapses. Co-culturing neurons with bone marrow-derived macrophages protected synapses against Aβ₄₂ fibrils; moreover, immune activation with glatiramer acetate (GA) conferred further protection against oligomers. Mechanisms involved increased Aβ₄₂ removal by macrophages, amplified by GA stimulation: fibrils were largely cleared through intracellular CD36/EEA1⁺-early endosomal proteolysis, while oligomers were primarily removed via extracellular/MMP-9 enzymatic degradation. In vivo studies in GA-immunized or CD115⁺-monocyte-grafted APP_(SWE)/PS1_(ΔE9)-transgenic mice followed by pre- and postsynaptic analyses of entorhinal cortex and hippocampal substructures corroborated our in vitro findings of macrophage-mediated synaptic preservation. Together, our data demonstrate that activated macrophages effectively clear Aβ₄₂ oligomers and rescue VGluT1/PSD95 synapses, providing rationale for harnessing macrophages to treat AD

Peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate Alzheimer-related disease.

Targeted overexpression of angiotensin-converting enzyme (ACE), an amyloid-β protein degrading enzyme, to brain resident microglia and peripheral myelomonocytes (ACE10 model) substantially diminished Alzheimer's-like disease in double-transgenic APPSWE/PS1ΔE9 (AD+) mice. In this study, we explored the impact of selective and transient angiotensin-converting enzyme overexpression on macrophage behaviour and the relative contribution of bone marrow-derived ACE10 macrophages, but not microglia, in attenuating disease progression. To this end, two in vivo approaches were applied in AD+ mice: (i) ACE10/GFP+ bone marrow transplantation with head shielding; and (ii) adoptive transfer of CD115+-ACE10/GFP+ monocytes to the peripheral blood. Extensive in vitro studies were further undertaken to establish the unique ACE10-macrophage phenotype(s) in response to amyloid-β1-42 fibrils and oligomers. The combined in vivo approaches showed that increased cerebral infiltration of ACE10 as compared to wild-type monocytes (∼3-fold increase; P < 0.05) led to reductions in cerebral soluble amyloid-β1-42, vascular and parenchymal amyloid-β deposits, and astrocytosis (31%, 47-80%, and 33%, respectively; P < 0.05-0.0001). ACE10 macrophages surrounded brain and retinal amyloid-β plaques and expressed 3.2-fold higher insulin-like growth factor-1 (P < 0.01) and ∼60% lower tumour necrosis factor-α (P < 0.05). Importantly, blood enrichment with CD115+-ACE10 monocytes in symptomatic AD+ mice resulted in pronounced synaptic and cognitive preservation (P < 0.05-0.001). In vitro analysis of macrophage response to well-defined amyloid-β1-42 conformers (fibrils, prion rod-like structures, and stabilized soluble oligomers) revealed extensive resistance to amyloid-β1-42 species by ACE10 macrophages. They exhibited 2-5-fold increased surface binding to amyloid-β conformers as well as substantially more effective amyloid-β1-42 uptake, at least 8-fold higher than those of wild-type macrophages (P < 0.0001), which were associated with enhanced expression of surface scavenger receptors (i.e. CD36, scavenger receptor class A member 1, triggering receptor expressed on myeloid cells 2, CD163; P < 0.05-0.0001), endosomal processing (P < 0.05-0.0001), and ∼80% increased extracellular degradation of amyloid-β1-42 (P < 0.001). Beneficial ACE10 phenotype was reversed by the angiotensin-converting enzyme inhibitor (lisinopril) and thus was dependent on angiotensin-converting enzyme catalytic activity. Further, ACE10 macrophages presented distinct anti-inflammatory (low inducible nitric oxide synthase and lower tumour necrosis factor-α), pro-healing immune profiles (high insulin-like growth factor-1, elongated cell morphology), even following exposure to Alzheimer's-related amyloid-β1-42 oligomers. Overall, we provide the first evidence for therapeutic roles of angiotensin-converting enzyme-overexpressing macrophages in preserving synapses and cognition, attenuating neuropathology and neuroinflammation, and enhancing resistance to defined pathognomonic amyloid-β forms

Resistance to receptor-blocking therapies primes tumors as targets for HER3-homing nanobiologics

Resistance to anti-tumor therapeutics is an important clinical problem. Tumor-targeted therapies currently used in the clinic are derived from antibodies or small molecules that mitigate growth factor activity. These have improved therapeutic efficacy and safety compared to traditional treatment modalities but resistance arises in the majority of clinical cases. Targeting such resistance could improve tumor abatement and patient survival. A growing number of such tumors are characterized by prominent expression of the human epidermal growth factor receptor 3 (HER3) on the cell surface. This study presents a “Trojan-Horse” approach to combating these tumors by using a receptor-targeted biocarrier that exploits the HER3 cell surface protein as a portal to sneak therapeutics into tumor cells by mimicking an essential ligand. The biocarrier used here combines several functions within a single fusion protein for mediating targeted cell penetration and non-covalent self-assembly with therapeutic cargo, forming HER3-homing nanobiologics. Importantly, we demonstrate here that these nanobiologics are therapeutically effective in several scenarios of resistance to clinically approved targeted inhibitors of the human EGF receptor family. We also show that such inhibitors heighten efficacy of our nanobiologics on naïve tumors by augmenting HER3 expression. This approach takes advantage of a current clinical problem (i.e. resistance to growth factor inhibition) and uses it to make tumors more susceptible to HER3 nanobiologic treatment. Moreover, we demonstrate a novel approach in addressing drug resistance by taking inhibitors against which resistance arises and re-introducing these as adjuvants, sensitizing tumors to the HER3 nanobiologics described here

The effects of enhancing angiotensin converting enzyme in myelomonocytes on ameliorating Alzheimer’s-related disease and preserving cognition

This review examines the role of angiotensin-converting enzyme (ACE) in the context of Alzheimer’s disease (AD) and its potential therapeutic value. ACE is known to degrade the neurotoxic 42-residue long alloform of amyloid β-protein (Aβ42), a peptide strongly associated with AD. Previous studies in mice, demonstrated that targeted overexpression of ACE in CD115+ myelomonocytic cells (ACE10 models) improved their immune responses to effectively reduce viral and bacterial infection, tumor growth, and atherosclerotic plaque. We further demonstrated that introducing ACE10 myelomonocytes (microglia and peripheral monocytes) into the double transgenic APPSWE/PS1ΔE9 murine model of AD (AD+ mice), diminished neuropathology and enhanced the cognitive functions. These beneficial effects were dependent on ACE catalytic activity and vanished when ACE was pharmacologically blocked. Moreover, we revealed that the therapeutic effects in AD+ mice can be achieved by enhancing ACE expression in bone marrow (BM)-derived CD115+ monocytes alone, without targeting central nervous system (CNS) resident microglia. Following blood enrichment with CD115+ ACE10-monocytes versus wild-type (WT) monocytes, AD+ mice had reduced cerebral vascular and parenchymal Aβ burden, limited microgliosis and astrogliosis, as well as improved synaptic and cognitive preservation. CD115+ ACE10-versus WT-monocyte-derived macrophages (Mo/MΦ) were recruited in higher numbers to the brains of AD+ mice, homing to Aβ plaque lesions and exhibiting a highly Aβ-phagocytic and anti-inflammatory phenotype (reduced TNFα/iNOS and increased MMP-9/IGF-1). Moreover, BM-derived ACE10-Mo/MΦ cultures had enhanced capability to phagocytose Aβ42 fibrils, prion-rod-like, and soluble oligomeric forms that was associated with elongated cell morphology and expression of surface scavenger receptors (i.e., CD36, Scara-1). This review explores the emerging evidence behind the role of ACE in AD, the neuroprotective properties of monocytes overexpressing ACE and the therapeutic potential for exploiting this natural mechanism for ameliorating AD pathogenesis

Osteopontin depletion in macrophages perturbs proteostasis via regulating UCHL1-UPS axis and mitochondria-mediated apoptosis

IntroductionOsteopontin (OPN; also known as SPP1), an immunomodulatory cytokine highly expressed in bone marrow-derived macrophages (BMMΦ), is known to regulate diverse cellular and molecular immune responses. We previously revealed that glatiramer acetate (GA) stimulation of BMMΦ upregulates OPN expression, promoting an anti-inflammatory, pro-healing phenotype, whereas OPN inhibition triggers a pro-inflammatory phenotype. However, the precise role of OPN in macrophage activation state is unknown.MethodsHere, we applied global proteome profiling via mass spectrometry (MS) analysis to gain a mechanistic understanding of OPN suppression versus induction in primary macrophage cultures. We analyzed protein networks and immune-related functional pathways in BMMΦ either with OPN knockout (OPNKO) or GA-mediated OPN induction compared with wild type (WT) macrophages. The most significant differentially expressed proteins (DEPs) were validated using immunocytochemistry, western blot, and immunoprecipitation assays.Results and discussionWe identified 631 DEPs in OPNKO or GA-stimulated macrophages as compared to WT macrophages. The two topmost downregulated DEPs in OPNKO macrophages were ubiquitin C-terminal hydrolase L1 (UCHL1), a crucial component of the ubiquitin-proteasome system (UPS), and the anti-inflammatory Heme oxygenase 1 (HMOX-1), whereas GA stimulation upregulated their expression. We found that UCHL1, previously described as a neuron-specific protein, is expressed by BMMΦ and its regulation in macrophages was OPN-dependent. Moreover, UCHL1 interacted with OPN in a protein complex. The effects of GA activation on inducing UCHL1 and anti-inflammatory macrophage profiles were mediated by OPN. Functional pathway analyses revealed two inversely regulated pathways in OPN-deficient macrophages: activated oxidative stress and lysosome-mitochondria-mediated apoptosis (e.g., ROS, Lamp1-2, ATP-synthase subunits, cathepsins, and cytochrome C and B subunits) and inhibited translation and proteolytic pathways (e.g., 60S and 40S ribosomal subunits and UPS proteins). In agreement with the proteome-bioinformatics data, western blot and immunocytochemical analyses revealed that OPN deficiency perturbs protein homeostasis in macrophages—inhibiting translation and protein turnover and inducing apoptosis—whereas OPN induction by GA restores cellular proteostasis. Taken together, OPN is essential for macrophage homeostatic balance via the regulation of protein synthesis, UCHL1-UPS axis, and mitochondria-mediated apoptotic processes, indicating its potential application in immune-based therapies

Resistance to receptor-blocking therapies primes tumors as targets for HER3-homing nanobiologics

Resistance to anti-tumor therapeutics is an important clinical problem. Tumor-targeted therapies currently used in the clinic are derived from antibodies or small molecules that mitigate growth factor activity. These have improved therapeutic efficacy and safety compared to traditional treatment modalities but resistance arises in the majority of clinical cases. Targeting such resistance could improve tumor abatement and patient survival. A growing number of such tumors are characterized by prominent expression of the human epidermal growth factor receptor 3 (HER3) on the cell surface. This study presents a “Trojan-Horse” approach to combating these tumors by using a receptor-targeted biocarrier that exploits the HER3 cell surface protein as a portal to sneak therapeutics into tumor cells by mimicking an essential ligand. The biocarrier used here combines several functions within a single fusion protein for mediating targeted cell penetration and non-covalent self-assembly with therapeutic cargo, forming HER3-homing nanobiologics. Importantly, we demonstrate here that these nanobiologics are therapeutically effective in several scenarios of resistance to clinically approved targeted inhibitors of the human EGF receptor family. We also show that such inhibitors heighten efficacy of our nanobiologics on naïve tumors by augmenting HER3 expression. This approach takes advantage of a current clinical problem (i.e. resistance to growth factor inhibition) and uses it to make tumors more susceptible to HER3 nanobiologic treatment. Moreover, we demonstrate a novel approach in addressing drug resistance by taking inhibitors against which resistance arises and re-introducing these as adjuvants, sensitizing tumors to the HER3 nanobiologics described here