Circulating extracellular vesicle-associated TGFß3 modulates response to cytotoxic therapy in head and neck squamous cell carcinoma.

Management of locally advanced head and neck squamous cell carcinoma (HNSCC) requires a multi-prong approach comprising surgery, radiation and/or chemotherapy, yet outcomes are limited. This is largely due to a paucity of biomarkers that can predict response to specific treatment modalities. Here, we evaluated TGFß3 protein levels in extracellular vesicles (EVs) released by HNSCC cells as a predictor for response to chemoradiation therapy (CRT). To this end, specific EV-fractions were isolated from cell lines or HNSCC patient plasma, and TGFß3 protein was quantified. In patients treated with CRT, TGFß3 levels were found to be significantly higher in plasma EV-fractions or non-responders compared with responders. High levels of TGFß3 levels in Annexin V-EVs were associated with the worst progression-free survival. In vitro experiments demonstrated that TGFß3 silencing sensitized HNSCC cells to cytotoxic therapies, and this phenotype could be rescued by treatment with exogenous. In addition, specific EV-fractions shed by cisplatin-resistant cells were sufficient to transfer the resistant phenotype to sensitive cells through activation of TGFß-signaling pathway. Therefore, our data show that TGFß3 transmitted through EV plays a significant role in response to cytotoxic therapy, which can be exploited as a potential biomarker for CRT response in HNSCC patients treated with curative intent.

A preliminary investigation of circulating extracellular vesicles and biomarker discovery associated with treatment response in head and neck squamous cell carcinoma.

BACKGROUND: There is a paucity of plasma-based biomarkers that prospectively segregate the outcome of patients with head and neck squamous-cell carcinoma (HNSCC) treated with chemoradiation therapy (CRT). Plasma extracellular vesicles (EVs) might be an alternative source for discovery of new specific markers present in patients with HNSCC, which could help to re-direct patients to appropriate curative therapies without delay. METHODS: In order to identify new markers in plasma compartments, Cholerae toxin B chain (CTB) and Annexin V (AV) were used to isolate EVs from pooled plasma samples from patients with locally advanced HNSCC who responded (CR, n = 6) or presented incomplete response (NR, n = 6) to CRT. The crude plasma and EVs cargo were screened by antibody array. RESULTS: Of the 370 polypeptides detected, 119 proteins were specific to NR patients while 38 were exclusive of the CR subjects. The Gene Set Enrichment Analysis (GSEA) and Search Tool for the Retrieval of Interacting Genes (STRING) database analysis indicated that the content of circulating plasma EVs might have a relevant function for the tumor intercellular communication in the HNSCC patients. CONCLUSION: This study provides a list of potential markers present in plasma compartments that might contribute to the development of tools for prediction and assessment of CRT response and potentially guide therapeutic decisions in this context.

Plasma biomarker discovery in preeclampsia using a novel differential isolation technology for circulating extracellular vesicles.

OBJECTIVE: To circumvent the complex protein milieu of plasma and discover robust predictive biomarkers for preeclampsia (PE), we investigate if phospholipid-binding ligands can reduce the milieu complexity by extracting plasma extracellular vesicles for biomarker discovery. STUDY DESIGN: Cholera toxin B chain (CTB) and annexin V (AV) which respectively binds GM1 ganglioside and phosphatidylserine were used to isolate extracellular vesicles from plasma of PE patients and healthy pregnant women. The proteins in the vesicles were identified using enzyme-linked immunosorbent assay, antibody array, and mass spectrometry. RESULTS: CTB and AV were found to bind 2 distinct groups of extracellular vesicles. Antibody array and enzyme-linked immunosorbent assay revealed that PE patients had elevated levels of CD105, interleukin-6, placental growth factor, tissue inhibitor of metallopeptidase 1, and atrial natriuretic peptide in cholera toxin B- but not AV-vesicles, and elevated levels of plasminogen activator inhibitor-1, pro-calcitonin, S100b, tumor growth factor ß, vascular endothelial growth factor receptor 1, brain natriuretic peptide, and placental growth factor in both cholera toxin B- and AV-vesicles. CD9 level was elevated in cholera toxin B-vesicles but reduced in AV vesicles of PE patients. Proteome analysis revealed that in cholera toxin B-vesicles, 87 and 222 proteins were present only in PE patients and healthy pregnant women respectively while in AV-vesicles, 104 and 157 proteins were present only in PE and healthy pregnant women, respectively. CONCLUSION: This study demonstrated for the first time that CTB and AV bind unique extracellular vesicles, and their protein cargo reflects the disease state of the patient. The successful use of these 2 ligands to isolate circulating plasma extracellular vesicles for biomarker discovery in PE represents a novel technology for biomarker discovery that can be applied to other specialties.

Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs.

BACKGROUND: Exosomes or secreted bi-lipid vesicles from human ESC-derived mesenchymal stem cells (hESC-MSCs) have been shown to reduce myocardial ischemia/reperfusion injury in animal models. However, as hESC-MSCs are not infinitely expansible, large scale production of these exosomes would require replenishment of hESC-MSC through derivation from hESCs and incur recurring costs for testing and validation of each new batch. Our aim was therefore to investigate if MYC immortalization of hESC-MSC would circumvent this constraint without compromising the production of therapeutically efficacious exosomes. METHODS: The hESC-MSCs were transfected by lentivirus carrying a MYC gene. The transformed cells were analyzed for MYC transgene integration, transcript and protein levels, and surface markers, rate of cell cycling, telomerase activity, karyotype, genome-wide gene expression and differentiation potential. The exosomes were isolated by HPLC fractionation and tested in a mouse model of myocardial ischemia/reperfusion injury, and infarct sizes were further assessed by using Evans' blue dye injection and TTC staining. RESULTS: MYC-transformed MSCs largely resembled the parental hESC-MSCs with major differences being reduced plastic adherence, faster growth, failure to senesce, increased MYC protein expression, and loss of in vitro adipogenic potential that technically rendered the transformed cells as non-MSCs. Unexpectedly, exosomes from MYC-transformed MSCs were able to reduce relative infarct size in a mouse model of myocardial ischemia/reperfusion injury indicating that the capacity for producing therapeutic exosomes was preserved. CONCLUSION: Our results demonstrated that MYC transformation is a practical strategy in ensuring an infinite supply of cells for the production of exosomes in the milligram range as either therapeutic agents or delivery vehicles. In addition, the increased proliferative rate by MYC transformation reduces the time for cell production and thereby reduces production costs.

Derivation and characterization of human fetal MSCs: an alternative cell source for large-scale production of cardioprotective microparticles.

The therapeutic effects of mesenchymal stem cells (MSCs) transplantation are increasingly thought to be mediated by MSC secretion. We have previously demonstrated that human ESC-derived MSCs (hESC-MSCs) produce cardioprotective microparticles in pig model of myocardial ischemia/reperfusion (MI/R) injury. As the safety and availability of clinical grade human ESCs remain a concern, MSCs from fetal tissue sources were evaluated as alternatives. Here we derived five MSC cultures from limb, kidney and liver tissues of three first trimester aborted fetuses and like our previously described hESC-derived MSCs; they were highly expandable and had similar telomerase activities. Each line has the potential to generate at least 10(16-19) cells or 10(7-10) doses of cardioprotective secretion for a pig model of MI/R injury. Unlike previously described fetal MSCs, they did not express pluripotency-associated markers such as Oct4, Nanog or Tra1-60. They displayed a typical MSC surface antigen profile and differentiated into adipocytes, osteocytes and chondrocytes in vitro. Global gene expression analysis by microarray and qRT-PCR revealed a typical MSC gene expression profile that was highly correlated among the five fetal MSC cultures and with that of hESC-MSCs (r(2)>0.90). Like hESC-MSCs, they produced secretion that was cardioprotective in a mouse model of MI/R injury. HPLC analysis of the secretion revealed the presence of a population of microparticles with a hydrodynamic radius of 50-65 nm. This purified population of microparticles was cardioprotective at approximately 1/10 dosage of the crude secretion.

High expression of MLANA in the plasma of patients with head and neck squamous cell carcinoma as a predictor of tumor progression.

BACKGROUND: There is a paucity of plasma-based biomarkers that predict outcome in patients with head and neck squamous cell carcinoma (HNSCC) treated with chemoradiation therapy (CRT). Here, we evaluate the prognostic potential of plasma Melanoma-Antigen Recognized by T-cells 1 (MLANA) in this setting. METHODS: MLANA expression in HNSCC lines were evaluated by reverse transcription polymerase chain reaction, whereas plasma levels were quantified using ELISA in 48 patients with locally advanced HNSCC undergoing a phase 2 trial with CRT. RESULTS: MLANA is expressed at variable levels in a panel of HNSCC lines. In plasma, levels were elevated in patients with tumor relapse compared to those without (P < .004); 73.9% of the patients expressing high plasma MLANA levels progressed with recurrent disease (P = .020). Multivariate analysis showed that plasma MLANA levels and tumor resectability were independent prognostic factors for progression free survival. CONCLUSION: Plasma MLANA expression appears to be an effective noninvasive biomarker for outcomes in patients treated with CRT, and could potentially guide therapeutic decisions in this context.

Extracellular vesicles yield predictive pre-eclampsia biomarkers.

Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-binding EVs were previously shown to be rich sources of biomarkers. Here we test if previously identified pre-eclampsia (PE) candidate biomarkers, TIMP-1 in CTB-EVs (CTB-TIMP) and PAI-1 in AV-EVs (AV-PAI) complement plasma PlGF in predicting PE in a low-risk obstetric population. Eight hundred and forty-three prospectively banked plasma samples collected at 28 + 0 to 32 + 0 gestation weeks in the Neonatal and Obstetrics Risk Assessment (NORA) cohort study were assayed by sandwich ELISAs for plasma PlGF, CTB-TIMP1 and AV-PAI1. Nineteen patients subsequently developed PE 7.3 (±2.9) weeks later at a mean gestational age of 36.1 ± 3.5 weeks. The biomarkers were assessed for their predictive accuracy for PE using stepwise multivariate logistic regression analysis with Firth correction and Areas under the curve (AUC). To achieve 100% sensitivity in predicting PE, the cut-off for plasma PlGF, CTB-TIMP1 & AV-PAI1 were set at <1235, ≤300 or >1300 and <10,550 pg/mL plasma, respectively. The corresponding AUCs, specificity and PPV at a 95% confidence interval were 0.92, 52.1% and 4.7%; 0.72, 44.5% and 4.0%; and 0.69, 21.5% and 2.9%, respectively. At 100% sensitivity, the three biomarkers had a combined AUC of 0.96, specificity of 78.6%, and PPV of 9.9%. This is the first large cohort validation of the utility of EV-associated analytes as disease biomarkers. Specifically, EV biomarkers enhanced the predictive robustness of an existing PE biomarker sufficiently to justify PE screening in a low-risk general obstetric population.

MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA.

Mesenchymal stem cell (MSC), a widely used adult stem cell candidate for regenerative medicine, has been shown to exert some of its therapeutic effects through the secretion of extracellular vesicles (EVs). These homogenously sized EVs of 100-150 ηm exhibited many exosome-like biophysical and biochemical properties and carry both proteins and RNAs. Recently, exosome-associated proteins in this MSC EV preparation were found to segregate primarily to those EVs that bind cholera toxin B chain (CTB), a GM1 ganglioside-specific ligand, and pulse-chase experiments demonstrated that these EVs have endosomal origin and carried many of the exosome-associated markers. Here, we report that only a fraction of the MSC EV proteome was found in CTB-bound EVs. Using Annexin V (AV) and Shiga toxin B subunit (ST) with affinities for phosphatidylserine and globotriaosylceramide, respectively, AV- and a ST-binding EV were identified. CTB-, AV- and ST-binding EVs all carried actin. However, the AV-binding EVs carried low or undetectable levels of the exosome-associated proteins. Only the ST-binding EVs carried RNA and EDA-containing fibronectin. Proteins in AV-binding EVs were also different from those released by apoptotic MSCs. CTB- and AV-binding activities were localized to the plasma membrane and cytoplasm of MSCs, while ST-binding activity was localized to the nucleus. Together, this study demonstrates that cells secrete many types of EVs. Specifically, MSCs secrete at least 3 types. They can be differentially isolated based on their affinities for membrane lipid-binding ligands. As the subcellular sites of the binding activities of these ligands and cargo load are different for each EV type, they are likely to have a different biogenesis pathway and possibly different functions.

Mesenchymal stem cells secrete immunologically active exosomes.

Mesenchymal stem cells (MSCs) have been shown to secrete exosomes that are cardioprotective. Here, we demonstrated that MSC exosome, a secreted membrane vesicle, is immunologically active. MSC exosomes induced polymyxin-resistant, MYD88-dependent secreted embryonic alkaline phosphatase (SEAP) expression in a THP1-Xblue, a THP-1 reporter cell line with an NFκB-SEAP reporter gene. In contrast to lipopolysaccharide, they induced high levels of anti-inflammatory IL10 and TGFß1 transcript at 3 and 72 h, and much attenuated levels of pro-inflammatory IL1B, IL6, TNFA and IL12P40 transcript at 3-h. The 3-h but not 72-h induction of cytokine transcript was abrogated by MyD88 deficiency. Primary human and mouse monocytes exhibited a similar exosome-induced cytokine transcript profile. Exosome-treated THP-1 but not MyD88-deficient THP-1 cells polarized activated CD4(+) T cells to CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) at a ratio of one exosome-treated THP-1 cell to 1,000 CD4(+) T cells. Infusion of MSC exosomes enhanced the survival of allogenic skin graft in mice and increased Tregs.

Mesenchymal stem cell: an efficient mass producer of exosomes for drug delivery.

Advances in biomedical research have generated an unprecedented number of potential targets for therapeutic intervention to treat disease or delay disease progression. Unfortunately, many of these targets are not druggable as they are intracellular, present in many cell types, poorly soluble or rapidly inactivated. Although synthetic drug vehicles have successfully circumvented many of these problems, natural particulates such as exosomes that intrinsically possess many attributes of a drug delivery vehicle are highly attractive as potentially better alternatives. Of the cell types known to produce exosomes, the readily available proliferative, immunosuppressive and clinically tested human mesenchymal stem cell (MSC) is the most prolific producer. Its exosomes are therapeutic in animal model of disease and exhibit immunosuppressive activity. The quality and quantity of exosome production is not compromised by immortalization to create a permanent MSC cell line. Therefore, MSC is well suited for mass production of exosomes that are ideal for drug delivery.

Therapeutic MSC exosomes are derived from lipid raft microdomains in the plasma membrane.

BACKGROUND: Mesenchymal stem cell (MSC) was previously shown to secrete lipid vesicles that when purified by high performance liquid chromatography as a population of homogenously sized particles with a hydrodynamic radius of 55-65 nm reduce infarct size in a mouse model of myocardial ischemia/reperfusion injury. As these vesicles exhibit many biophysical and biochemical properties of exosomes, they were identified as exosomes. Here we investigated if these lipid vesicles were indeed exosomes that have an endosomal biogenesis. METHOD: In most cells, endocytosis is thought to occur at specialized microdomains known as lipid rafts. To demonstrate an endosomal origin for MSC exosomes, MSCs were pulsed with ligands e.g. transferrin (Tfs) and Cholera Toxin B (CTB) that bind receptors in lipid rafts. The endocytosed ligands were then chased to determine if they were incorporated into the exosomes. RESULTS: A fraction of exogenous Tfs was found to recycle into MSC exosomes. When MSCs were pulsed with labelled Tfs in the presence of chlorpromazine, an inhibitor of clathrin-mediated endocytosis, Tf incorporation in CD81-immunoprecipitate was reduced during the chase. CTB which binds GM1 gangliosides that are enriched in lipid rafts extracted exosome-associated proteins, CD81, CD9, Alix and Tsg101 from MSC-conditioned medium. Exogenous CTBs were pulse-chased into secreted vesicles. Extraction of Tf- or CTB-binding vesicles in an exosome preparation mutually depleted each other. Inhibition of sphingomyelinases reduced CTB-binding vesicles. CONCLUSION: Together, our data demonstrated that MSC exosomes are derived from endocytosed lipid rafts and that their protein cargo includes exosome-associated proteins CD81, CD9, Alix and Tsg101.

Proteolytic Potential of the MSC Exosome Proteome: Implications for an Exosome-Mediated Delivery of Therapeutic Proteasome.

Mesenchymal stem cells (MSCs) are used in many of the current stem cell-based clinical trials and their therapeutic efficacy has increasingly been attributed to secretion of paracrine factors. We have previously demonstrated that a therapeutic constituent of this secretion is exosome, a secreted bilipid membrane vesicle of ~50-100 nm with a complex cargo that is readily internalized by H9C2 cardiomyocytes. It reduces infarct size in a mouse model of myocardial ischemia/reperfusion (MI/R) injury. We postulate that this therapeutic efficacy is derived from the synergy of a select permutation of individual exosome components. To identify protein candidates in this permutation, the proteome was profiled and here we identified 20S proteasome as a protein candidate. Mass spectrometry analysis detected all seven α and seven ß chains of the 20S proteasome, and also the three beta subunits of "immunoproteasome" with a very high confidence level. We demonstrated that a functional proteasome copurified with MSC exosomes with a density of 1.10-1.18 g/mL, and its presence correlated with a modest but significant reduction in oligomerized protein in a mouse model of myocardial infarction. Circulating proteasomes in human blood also copurified with exosomes. Therefore, 20S proteasome is a candidate exosome protein that could synergize with other constituents to ameliorate tissue damage.

Delineating biological pathways unique to embryonic stem cell-derived insulin-producing cell lines from their noninsulin-producing progenitor cell lines.

To identify unique biochemical pathways in embryonic stem cell-derived insulin-producing cells as potential therapeutic targets to prevent or delay beta-cell dysfunction or death in diabetic patients, comparative genome-wide gene expression studies of recently derived mouse insulin-producing cell lines and their progenitor cell lines were performed using microarray technology. Differentially expressed genes were functionally clustered to identify important biochemical pathways in these insulin-producing cell lines. Biochemical or cellular assays were then performed to assess the relevance of these pathways to the biology of these cells. A total of 185 genes were highly expressed in the insulin-producing cell lines, and computational analysis predicted the pentose phosphate pathway (PPP), clathrin-mediated endocytosis, and the peroxisome proliferator-activated receptor (PPAR) signaling pathway as important pathways in these cell lines. Insulin-producing ERoSHK cells were more resistant to hydrogen peroxide (H(2)O(2))-induced oxidative stress. Inhibition of PPP by dehydroepiandrosterone and 6-aminonicotinamide abrogated this H(2)O(2) resistance with a concomitant decrease in PPP activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Clathrin-mediated endocytosis, which is essential in maintaining membrane homeostasis in secreting cells, was up-regulated by glucose in ERoSHK but not in their progenitor ERoSH cells. Its inhibition by chlorpromazine at high glucose concentration was toxic to the cells. Troglitazone, a PPARG agonist, up-regulated expression of Ins1 and Ins2 but not Glut2. Gene expression analysis has identified the PPP, clathrin-mediated endocytosis, and the PPAR signaling pathway as the major delineating pathways in these insulin-producing cell lines, and their biological relevance was confirmed by biochemical and cellular assays.