Exposure to environmental airborne particulate matter caused wide-ranged transcriptional changes and accelerated Alzheimer's-related pathology: A mouse study.

Air pollution poses a significant threat to human health, though a clear understanding of its mechanism remains elusive. In this study, we sought to better understand the effects of various sized particulate matter from polluted air on Alzheimer's disease (AD) development using an AD mouse model. We exposed transgenic Alzheimer's mice in their prodromic stage to different sized particulate matter (PM), with filtered clean air as control. After 3 or 6 months of exposure, mouse brains were harvested and analyzed. RNA-seq analysis showed that various PM have differential effects on the brain transcriptome, and these effects seemed to correlate with PM size. Many genes and pathways were affected after PM exposure. Among them, we found a strong activation in mRNA Nonsense Mediated Decay pathway, an inhibition in pathways related to transcription, neurogenesis and survival signaling as well as angiogenesis, and a dramatic downregulation of collagens. Although we did not detect any extracellular Aß plaques, immunostaining revealed that both intracellular Aß1-42 and phospho-Tau levels were increased in various PM exposure conditions compared to the clean air control. NanoString GeoMx analysis demonstrated a remarkable activation of immune responses in the PM exposed mouse brain. Surprisingly, our data also indicated a strong activation of various tumor suppressors including RB1, CDKN1A/p21 and CDKN2A/p16. Collectively, our data demonstrated that exposure to airborne PM caused a profound transcriptional dysregulation and accelerated Alzheimer's-related pathology.

ß-Amyloid targeting nanodrug for neuron-specific delivery of nucleic acids in Alzheimer's disease mouse models.

Delivery of therapeutic substances into the brain poses a significant challenge in the treatment of neurological disorders. This is primarily due to the blood-brain barrier (BBB), which restricts access, alongside the limited stability and distribution of these agents within the brain tissue. Here we demonstrate an efficient delivery of microRNA (miRNA) and antisense RNA preferentially to neurons compared to astroglia in the brain of healthy and Alzheimer's disease mice, via disulfide-linked conjugation with poly(ß-L-malic acid-trileucine)-copolymer a biodegradable, amphiphilic, and multivalent platform. By conjugating a D-configured (D3)-peptide (vector) for specific targeting, highly efficient delivery across the BBB is achieved through the Low-Density Lipoprotein Receptor-Related Protein-1 (LRP-1) transcytosis pathway, amyloid beta (Aß) peptides. Nanodrug distribution was determined by fluorescent labeling and analyzed by microscopy in neurons, astroglia, and in extracellular amyloid plaques typical for Alzheimer's disease. Whereas D-configured BBB-vectors can efficiently target neurons, L-configured (e.g., AP2-peptide) guided vector can only cross BBB but not seem to bind neurons. An analysis of post-injection fluorescence distribution, and RNA-seq followed by real-time PCR validation, confirmed a successful in vivo delivery of morpholino-miRNA-186 nanoconjugates into mouse brain. The size and fluorescence intensity of the intracellular nanodrug particulates were analyzed and verified by a competition with non-fluorescent conjugates. Differentially expressed genes (DEGs) from RNA-seq were identified in the nanodrug injected mice, and the changes of selected DEGs related to Alzheimer's disease were further validated by western blot and real-time PCR. Collectively, these results demonstrated that D3-peptide-conjugated nanopolymer drug is able to achieve neuron-selective delivery of miRNA and can serve as an efficient brain delivery vehicle in Alzheimer's disease (AD) mouse models.

Signature Effects of Vector-Guided Systemic Nano Bioconjugate Delivery Across Blood-Brain Barrier of Normal, Alzheimer's, and Tumor Mouse Models.

The ability to cross the blood-brain barrier (BBB) is critical for targeted therapy of the central nerve system (CNS). Six peptide vectors were covalently attached to a 50 kDa poly(ß-l-malic acid)-trileucine polymer forming P/LLL(40%)/vector conjugates. The vectors were Angiopep-2 (AP2), B6, Miniap-4 (M4), and d-configurated peptides D1, D3, and ACI-89, with specificity for transcytosis receptors low-density lipoprotein receptor-related protein-1 (LRP-1), transferrin receptor (TfR), bee venom-derived ion channel, and Aß/LRP-1 related transcytosis complex, respectively. The BBB-permeation efficacies were substantially increased ("boosted") in vector conjugates of P/LLL(40%). We have found that the copolymer group binds at the endothelial membrane and, by an allosterically membrane rearrangement, exposes the sites for vector-receptor complex formation. The specificity of vectors is indicated by competition experiments with nonconjugated vectors. P/LLL(40%) does not function as an inhibitor, suggesting that the copolymer binding site is eliminated after binding of the vector-nanoconjugate. The two-step mechanism, binding to endothelial membrane and allosteric exposure of transcytosis receptors, is supposed to be an integral feature of nanoconjugate-transcytosis pathways. In vivo brain delivery signatures of the nanoconjugates were recapitulated in mouse brains of normal, tumor (glioblastoma), and Alzheimer's disease (AD) models. BBB permeation of the tumor was most efficient, followed by normal and then AD-like brain. In tumor-bearing and normal brains, AP2 was the top performing vector; however, in AD models, D3 and D1 peptides were superior ones. The TfR vector B6 was equally efficient in normal and AD-model brains. Cross-permeation efficacies are manifested through modulated vector coligation and dosage escalation such as supra-linear dose dependence and crossover transcytosis activities.

Author Correction: Blood-brain barrier permeable nano immunoconjugates induce local immune responses for glioma therapy.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20129-9.

Author Correction: Blood-brain barrier permeable nano immunoconjugates induce local immune responses for glioma therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Blood-brain barrier permeable nano immunoconjugates induce local immune responses for glioma therapy.

Brain glioma treatment with checkpoint inhibitor antibodies to cytotoxic T-lymphocyte-associated 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(ß-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.

Polymalic acid chlorotoxin nanoconjugate for near-infrared fluorescence guided resection of glioblastoma multiforme.

Maximal surgical resection of glioma remains the single most effective treatment. Tools to guide the resection while avoiding removal of normal brain tissues can aid surgeons in achieving optimal results. One strategy to achieve this goal is to rely upon interoperative fluorescence staining of tumor cells in vivo, that can be visualized by the surgeon during resection. Towards this goal we have designed a biodegradable fluorescent mini nano imaging agent (NIA) with high specificity for U87MG glioma cells and previously unmet high light emission. The NIA is the conjugate of polymalic acid (PMLA) with chlorotoxin for tumor targeting, indocyanine green (ICG) for NIR fluorescence and the tri-leucin peptide as fluorescence enhancer. PMLA as a multivalent platform carries several molecules of ICG and the other ligands. The NIA recognizes multiple sites on glioma cell surface, demonstrated by the effects of single and combined competitors. Systemic IV injection into xenogeneic mouse model carrying human U87MG glioblastoma indicated vivid tumor cell binding and internalization of NIA resulting in intensive and long-lasting tumor fluorescence. The NIA is shown to greatly improve tumor removal supporting its utility in clinical applications.

A Combination of Tri-Leucine and Angiopep-2 Drives a Polyanionic Polymalic Acid Nanodrug Platform Across the Blood-Brain Barrier.

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 ß-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.

Coarse particulate matter (PM2.5-10) in Los Angeles Basin air induces expression of inflammation and cancer biomarkers in rat brains.

Air pollution is linked to brain inflammation, which accelerates tumorigenesis and neurodegeneration. The molecular mechanisms that connect air pollution with brain pathology are largely unknown but seem to depend on the chemical composition of airborne particulate matter (PM). We sourced ambient PM from Riverside, California, and selectively exposed rats to coarse (PM2.5-10: 2.5-10 µm), fine (PM<2.5: <2.5 µm), or ultrafine particles (UFPM: <0.15 µm). We characterized each PM type via atomic emission spectroscopy and detected nickel, cobalt and zinc within them. We then exposed rats separately to each PM type for short (2 weeks), intermediate (1-3 months) and long durations (1 year). All three metals accumulated in rat brains during intermediate-length PM exposures. Via RNAseq analysis we then determined that intermediate-length PM2.5-10 exposures triggered the expression of the early growth response gene 2 (EGR2), genes encoding inflammatory cytokine pathways (IL13-Rα1 and IL-16) and the oncogene RAC1. Gene upregulation occurred only in brains of rats exposed to PM2.5-10 and correlated with cerebral nickel accumulation. We hypothesize that the expression of inflammation and oncogenesis-related genes is triggered by the combinatorial exposure to certain metals and toxins in Los Angeles Basin PM2.5-10.

Proteomic Analysis Reveals Autophagy as Pro-Survival Pathway Elicited by Long-Term Exposure with 5-Azacitidine in High-Risk Myelodysplasia.

Azacytidine (5-AZA) is the standard first-choice treatment for high-risk myelodysplasia (MDS) patients. However, the clinical outcome for those patients who interrupt treatment or whose disease failed to respond is very poor. In order to identify the cellular pathways that are modified by long-term exposure to 5-AZA, we evaluated key proteins associated with the autophagy pathway by reverse-phase microarray (RPPA). Comparing bone marrow mononucleated cells (BMMCs) obtained from 20 newly-diagnosed patients and after four 5-AZA cycles we found an increased autophagy signaling. We then evaluated ex-vivo the effect of the combination of 5-AZA with autophagy inhibitors chloroquine (CQ) and leupeptin. Since 5-AZA and CQ showed synergism due to an increase of basal autophagy after 5-AZA exposure, we adopted a sequential treatment treating BMMCs with 5 µM 5-AZA for 72 h followed by 10 µM CQ for 24 h and found increased apoptosis, associated to a reduction of G2M phase and increase in G0-G1 phase. Long-term exposure to 5-AZA induced the reduction of the autophagic marker SQSTM1/p62, reversible by CQ or leupeptin exposure. In conclusion, we identified autophagy as a compensatory pathway occurring in MDS-BM after long-term exposure to 5-AZA and we provided evidences that a sequential treatment of 5-AZA followed by CQ could improve 5-AZA efficacy, providing novel insight for tailored therapy in MDS patients progressing after 5-AZA therapy.

Murine models of breast cancer bone metastasis.

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 in vivo and ex vivo 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.

Normalization of Reverse Phase Protein Microarray Data: Choosing the Best Normalization Analyte.

Reverse phase protein microarray (RPMA) are a relatively recent but widely used approach to measure a large number of proteins, in their original and posttranslational modified forms, in a small clinical sample. Data normalization is fundamental for this technology, to correct for the sample-to-sample variability in the many possible confounding factors: extracellular proteins, red blood cells, different number of cells in the sample. To address this need, we adopted gene microarray algorithms to tailor the RPMA processing and analysis to the specific study set. Using geNorm and NormFinder algorithms, we screened seven normalization analytes (ssDNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), α/ß-tubulin, mitochondrial ribosomal protein L11 (MRPL11), ribosomal protein L13a (RPL13a), ß-actin, and total protein) across different sample sets, including cell lines, blood contaminated tissues, and tissues subjected to laser capture microdissection (LCM), to identify the analyte with the lowest variability. Specific normalization analytes were found to be advantageous for different classes of samples, with ssDNA being the optimal analyte to normalize blood contaminated samples.

Excess TGF-ß mediates muscle weakness associated with bone metastases in mice.

Cancer-associated muscle weakness is a poorly understood phenomenon, 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 transforming growth factor (TGF)-ß, 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 and calcium (Ca(2+)) release channel (RyR1). The oxidized RyR1 channels leaked Ca(2+), resulting in lower intracellular signaling, which is required for proper muscle contraction. We found that inhibiting RyR1 leakage, TGF-ß signaling, TGF-ß release from bone or Nox4 activity improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, increased Nox4 binding to RyR1 and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a nonmalignant metabolic bone disorder associated with increased TGF-ß activity. Thus, pathological TGF-ß release from bone contributes to muscle weakness by decreasing Ca(2+)-induced muscle force production.

Mapping protein signal pathway interaction in sarcoma bone metastasis: linkage between rank, metalloproteinases turnover and growth factor signaling pathways.

We applied reverse phase protein microarrays technology to map signal pathway interactions in a discovery set of 34 soft tissue sarcoma (STS) bone metastases compared to healthy bone. Proteins associated with matrix remodeling (MMP), adhesion (FAK Y576/577, Syndecan-1), and growth/survival (IGF1R Y1135/1136, PI3K, EGFR) were elevated in metastasis compared to normal bone. Linkage between Syndecan-1, FAK Y576/577, Shc Y317, and EGFR, IGF Y1135/1136, PI3K/AKT was a prominent feature of STS bone metastasis. Elevated linkage between RANKL and 4EBP1 T37/46, EGFR, IGF1R Y1135/1136, Src Y41, Shc Y317, PI3Kp110γ was associated with short survival. Finally, we tested the hypothesis that signal pathway proteins augmented in the STS bone metastasis may provide clues to understand the subset of primary STS that metastasize. The most representative molecules identified in the discovery set were validated on an independent series of 82 primary STS by immunohistochemistry applied to a tissue microarray. The goal was to correlate the molecular profile in the primary tumors with a higher likelihood of metastasis. Elevation of activated kinase substrate endpoints IRS1 S612, 4EBP1 T37/46, FAK Y576/577 and loss of Fibronectin, were associated with a higher likelihood of metastases. These data indicate that the linkage between matrix remodeling, adhesion, and growth signaling may drive STS metastasis and can be the basis for prognostic and therapeutic strategies.

Retinal pigment epithelium (RPE) exosomes contain signaling phosphoproteins affected by oxidative stress.

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 (Plafker, Adv. Exp. Med. Biol. 664 (2010) 447-56; Qin, Drug Dev. Res. 68 (2007) 213-225). 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 (Davuluri et al., Arch. Ophthalmol. 127 (2009) 613-21). 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. Seventy two proteins were shown to be released by ARPE-19 cells and compartmentalized within exosomes. Forty one 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., AMPKα1 (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.

Elevated TNFR1 and serotonin in bone metastasis are correlated with poor survival following bone metastasis diagnosis for both carcinoma and sarcoma primary tumors.

PURPOSE: There is an urgent need for therapies that will reduce the mortality of patients with bone metastasis. In this study, we profiled the protein signal pathway networks of the human bone metastasis microenvironment. The goal was to identify sets of interacting proteins that correlate with survival time following the first diagnosis of bone metastasis. EXPERIMENTAL DESIGN: Using Reverse Phase Protein Microarray technology, we measured the expression of 88 end points in the bone microenvironment of 159 bone metastasis tissue samples derived from patients with primary carcinomas and sarcomas. RESULTS: Metastases originating from different primary tumors showed similar levels of cell signaling across tissue types for the majority of proteins analyzed, suggesting that the bone microenvironment strongly influences the metastatic tumor signaling profiles. In a training set (72 samples), TNF receptor 1, alone (P = 0.0013) or combined with serotonin (P = 0.0004), TNFα (P = 0.0214), and RANK (P = 0.0226), was associated with poor survival, regardless of the primary tumor of origin. Results were confirmed by (i) analysis of an independent validation set (71 samples) and (ii) independent bioinformatic analysis using a support vector machine learning model. Spearman rho analysis revealed a highly significant number of interactions intersecting with ERα S118, serotonin, TNFα, RANKL, and matrix metalloproteinase in the bone metastasis signaling network, regardless of the primary tumor. The interaction network pattern was significantly different in the short versus long survivors. CONCLUSIONS: TNF receptor 1 and neuroendocrine-regulated protein signal pathways seem to play an important role in bone metastasis and may constitute a novel drug-targetable mechanism of seed-soil cross talk in bone metastasis.

Role of TGF-ß in breast cancer bone metastases.

Breast cancer is the most prevalent cancer among females worldwide leading to approximately 350,000 deaths each year. It has long been known that cancers preferentially metastasize to particular organs, and bone metastases occur in ~70% of patients with advanced breast cancer. Breast cancer bone metastases are predominantly osteolytic and accompanied by increased fracture risk, pain, nerve compression and hypercalcemia, causing severe morbidity. In the bone matrix, transforming growth factor-ß (TGF-ß) is one of the most abundant growth factors, which is released in active form upon tumor-induced osteoclastic bone resorption. TGF-ß, in turn, stimulates bone metastatic tumor cells to secrete factors that further drive osteolytic bone destruction adjacent to the tumor. Thus, TGF-ß is a crucial factor responsible for driving the feed-forward vicious cycle of cancer growth in bone. Moreover, TGF-ß activates epithelial-to-mesenchymal transition, increases tumor cell invasiveness and angiogenesis and induces immunosuppression. Blocking the TGF-ß signaling pathway to interrupt this vicious cycle between breast cancer and bone offers a promising target for therapeutic intervention to decrease skeletal metastasis. This review will describe the role of TGF-ß in breast cancer and bone metastasis, and pre-clinical and clinical data will be evaluated for the potential use of TGF-ß inhibitors in clinical practice to treat breast cancer bone metastases.

Improved data normalization methods for reverse phase protein microarray analysis of complex biological samples.

Reverse phase protein microarrays (RPMA) are designed for quantitative, multiplexed analysis of proteins, and their posttranslational modified forms, from a limited amount of sample. To correct for sample to sample variability due to the number of cells in each lysate and the presence of extracellular proteins or red blood cells, a normalization method is required that is independent of these potentially confounding parameters. We adopted a gene microarray algorithm for use with RPMA to optimize the proteomic data normalization process and developed a systematic approach to RPMA processing and analysis, tailored to the study set. Our approach capitalizes on the gene microarray algorithms geNorm and NormFinder to identify the normalization parameter with the lowest variability across a proteomic sample set. Seven analytes (ssDNA, glyceraldehyde 3-phosphate dehydrogenase, α/ß-tubulin, mitochondrial ribosomal protein L11, ribosomal protein L13a, ß-actin, and total protein) were compared across sample sets including cell lines, tissues subjected to laser capture microdissection, and blood-contaminated tissues. We examined normalization parameters to correct for red blood cell content. We show that single-stranded DNA (ssDNA) is proportional to total non-red blood cell content and is a suitable RPMA normalization parameter. Simple modifications to RPMA processing allow flexibility in using ssDNA-or protein-based normalization molecules.

Identification of potential biomarkers for giant cell tumor of bone using comparative proteomics analysis.

Giant cell tumor of bone can be locally aggressive and occasionally can metastasize in the lungs. To identify new markers predictive of aggressive behavior, we analyzed five patients who developed lung metastasis and five who remained disease free for a minimum of 5 years. Using two-dimensional electrophoresis, we detected 28 differentially expressed spots. Fourteen spots were identified using mass spectrometry, including seven up-regulated and seven down-regulated in metastatic samples and classified according to functional categories. We then selected five proteins involved in cell cycle or apoptosis. Thioredoxin peroxidase, allograft inflammatory factor 1, and ubiquitin E2N had more than threefold up-regulation; glutathione peroxidase 1 had 1.9-fold up-regulation; and heat shock protein 27 showed down-regulation in metastatic samples with a very low P value. After validation and analysis of protein levels, evaluation of clinical impact was assessed in a much wider cohort of primary archival specimens. Immunodetection showed a higher frequency of thioredoxin peroxidase, allograft inflammatory factor 1, ubiquitin E2N, and glutathione peroxidase 1 overexpression in primary tumors that developed into lung metastases or that locally relapsed than in the disease-free group, with variable stain intensity and distribution. Kaplan-Meier analysis showed that high expression of glutathione peroxidase 1 was strongly related to local recurrence and metastasis, suggesting that its up-regulation may identify a subset of high-risk patients with giant cell tumor prone to receive diverse clinical management.

Differential gene expression in classic giant cell tumours of bone: Tenascin C as biological risk factor for local relapses and metastases.

AIMS: To identify candidate prognostic biological markers useful in selecting high-risk patients with classic primary giant cell tumours (GCT). GCT specimens with different behaviour associated with an increased risk of local and/or distant relapses were studied. METHODS AND RESULTS: Screening mRNA microarray analysis followed by real-time polymerase chain reaction and immunohistochemistry on tissue microarray sections was used to validate the prognostic role of differentially expressed genes on a larger series by statistical analysis tests. Global gene expression profiling identified 109 differentially expressed genes according to prognosis. A correlation was found between mRNA levels and clinical outcome identifying Tenascin C (TNC) as the most significant prognostic biological marker. By means of backward Wald logistic regression, TNC overexpression defined an eightfold increased risk for metastasis and fourfold for local recurrence. At the protein level, TNC immunoreactivity resulted in a significant difference in the disease-free survival probability curves, providing a stratification for GCT patients, useful for predicting relapse. CONCLUSIONS: Our study provides important data for the selection of biomarkers with a significant clinical impact and suggests the importance of TNC expression in identifying GCT patients at a higher risk of relapse for closer follow-up and adjuvant medical therapy.