Cancer-associated muscle weakness is poorly understood and there is no effective treatment. Here, we find that seven different mouse models of human osteolytic bone metastases, representing breast, lung and prostate cancers, as well as multiple myeloma exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that TGF-β, released from the bone surface as a result of metastasis-induced bone destruction upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor/calcium (Ca2+) release channel (RyR1). The oxidized RyR1 channels leaked Ca2+, resulting in lower intracellular signaling required for proper muscle contraction. We found that inhibiting RyR1 leak, TGF-β signaling, TGF-β release from bone or Nox4 all improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast cancer- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, higher levels of Nox4 protein and Nox4 binding to RyR1, and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a non-malignant metabolic bone disorder associated with increased TGF-β activity. Thus, metastasis-induced TGF-β release from bone contributes to muscle weakness by decreasing Ca2+-induced muscle force production.

Brain glioma treatment with checkpoint inhibitor antibodies to cytotoxic T-lymphocyteassociated antigen 4 (a-CTLA-4) and programmed cell death-1 (a-PD-1) was largely unsuccessful due to their inability to cross blood-brain barrier (BBB). Here we describe targeted nanoscale immunoconjugates (NICs) on natural biopolymer scaffold, poly(β-L-malic acid), with covalently attached a-CTLA-4 or a-PD-1 for systemic delivery across the BBB and activation of local brain anti-tumor immune response. NIC treatment of mice bearing intracranial GL261 glioblastoma (GBM) results in an increase of CD8+ T cells, NK cells and macrophages with a decrease of regulatory T cells (Tregs) in the brain tumor area. Survival of GBM-bearing mice treated with NIC combination is significantly longer compared to animals treated with single checkpoint inhibitor-bearing NICs or free a-CTLA-4 and a-PD-1. Our study demonstrates trans-BBB delivery of tumor-targeted polymer-conjugated checkpoint inhibitors as an effective GBM treatment via activation of both systemic and local privileged brain tumor immune response.

One of the major problems facing the treatment of neurological disorders is the poor delivery of therapeutic agents into the brain. Our goal is to develop a multifunctional and biodegradable nanodrug delivery system that crosses the blood–brain barrier (BBB) to access brain tissues affected by neurological disease. In this study, we synthesized a biodegradable nontoxic β-poly­(l-malic acid) (PMLA or P) as a scaffold to chemically bind the BBB crossing peptides Angiopep-2 (AP2), MiniAp-4 (M4), and the transferrin receptor ligands cTfRL and B6. In addition, a trileucine endosome escape unit (LLL) and a fluorescent marker (rhodamine or rh) were attached to the PMLA backbone. The pharmacokinetics, BBB penetration, and biodistribution of nanoconjugates were studied in different brain regions and at multiple time points via optical imaging. The optimal nanoconjugate, P/LLL/AP2/rh, produced significant fluorescence in the parenchyma of cortical layers II/III, the midbrain colliculi, and the hippocampal CA1-3 cellular layers 30 min after a single intravenous injection; clearance was observed after 4 h. The nanoconjugate variant P/LLL/rh lacking AP2, or the variant P/AP2/rh lacking LLL, showed significantly less BBB penetration. The LLL moiety appeared to stabilize the nanoconjugate, while AP2 enhanced BBB penetration. Finally, nanoconjugates containing the peptides M4, cTfRL, and B6 displayed comparably little and/or inconsistent infiltration of brain parenchyma, likely due to reduced trans-BBB movement. P/LLL/AP2/rh can now be functionalized with intra-brain targeting and drug treatment moieties that are aimed at molecular pathways implicated in neurological disorders.

Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness among the elderly population in the industrialized world. One of the typical features of this pathology is the gradual death of retinal pigment epithelial (RPE) cells, which are essential for maintaining photoreceptor functions and survival. The etiology is multifactorial, and oxidative stress is clearly one of the key factors involved in disease pathogenesis. Recent work has revealed the presence of phosphorylated signaling proteins in the vitreous humour of patients affected by AMD or other retinal diseases. While the location of these signaling proteins is typically the cell membrane or intracellular compartments, vitreous samples were proven to be cell-free. To gain a better understanding of how these proteins can be shed into the vitreous, we used Reverse Phase Protein Arrays (RPMA) to analyze the protein and phosphoprotein content of exosomes shed by cultured ARPE-19 cells under oxidative stress conditions. 72 proteins were shown to be released by ARPE-19 cells and compartmentalized within exosomes. 41 of them were selectively detected in their post-translationally modified form (i.e., phosphorylated or cleaved) for the first time in exosomes. Sets of these proteins were linked together reflecting activation of pathway units within exosomes. A subset of (phospho)proteins were altered in exosomes secreted by ARPE-19 cells subjected to oxidative stress, compared to that secreted by control/non stressed cells. Stress-altered exosome proteins were found to be involved in pathways regulating apoptosis/survival (i.e, Bak, Smac/Diablo, PDK1 (S241), Akt (T308), Src (Y416), Elk1 (S383), ERK 1/2 (T202/Y204)) and cell metabolism (i.e., AMPK α1 (S485), Acetyl-CoA carboxylase (S79), LDHA). Exosomes may thus represent the conduit through which membrane and intracellular signaling proteins are released into the vitreous. Changes in their (phospho)protein content upon stress conditions suggest their possible role in mediating cell-cell signaling during physio-pathological events; furthermore, exosomes may represent a potential source of biomarkers.

Bone metastases cause significant morbidity and mortality in late-stage breast cancer patients and are currently considered incurable. Investigators rely on translational models to better understand the pathogenesis of skeletal complications of malignancy in order to identify therapeutic targets that may ultimately prevent and treat solid tumor metastasis to bone. Many experimental models of breast cancer bone metastases are in use today, each with its own caveats. In this methods review, we characterize the bone phenotype of commonly utilized human- and murine-derived breast cell lines that elicit osteoblastic and/or osteolytic destruction of bone in mice and report methods for optimizing tumor-take in murine models of bone metastasis. We then provide protocols for four of the most common xenograft and syngeneic inoculation routes for modeling breast cancer metastasis to the skeleton in mice, including the intra-cardiac, intra-arterial, orthotopic and intra-tibial methods of tumor cell injection. Recommendations for and assessment of tumor progression and bone destruction are provided, followed by discussion of the strengths and limitations of the available tools and translational models that aid investigators in the study of breast cancer metastasis to bone.