Michael W. Graner

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Mutant Epidermal Growth Factor Receptor (EGFRvIII) Contributes to Head and Neck Cancer Growth and Resistance to EGFR Targeting

Purpose: Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinoma (HNSCC) where expression levels correlate with decreased survival. Therapies that block EGFR have shown limited efficacy in clinical trials and primarily when combined with standard therapy. The most common form of mutant EGFR (EGFRvIII) has been described in several cancers, chiefly glioblastoma. The present study was undertaken to determine the incidence of EGFRvIII expression in HNSCC and the biological consequences of EGFRvIII on tumor growth in response to EGFR targeting. Experimental Design: Thirty-three HNSCC tumors were evaluated by immunostaining and reverse transcription-PCR for EGFRvIII expression. A representative HNSCC cell line was stably transfected with an EGFRvIII expression construct. EGFRvIII-expressing cells and vector-transfected controls were compared for growth rates in vitro and in vivo as well as chemotherapy-induced apoptosis and the consequences of EGFR inhibition using the chimeric monoclonal antibody C225/cetuximab/Erbitux. Results: EGFRvIII expression was detected in 42% of HNSCC tumors where EGFRvIII was always found in conjunction with wild-type EGFR. HNSCC cells expressing EGFRvIII showed increased proliferation in vitro and increased tumor volumes in vivo compared with vector-transfected controls. Furthermore, EGFRvIII-transfected HNSCC cells showed decreased apoptosis in response to cisplatin and decreased growth inhibition following treatment with C225 compared with vector-transfected control cells. Conclusions: EGFRvIII is expressed in HNSCC where it contributes to enhanced growth and resistance to targeting wild-type EGFR. The antitumor efficacy of EGFR targeting strategies may be enhanced by the addition of EGFRvIII-specific blockade.

Proteomic and immunologic analyses of brain tumor exosomes

Brain tumors are horrific diseases with almost universally fatal outcomes; new therapeutics are desperately needed and will come from improved understandings of glioma biology. Exosomes are endo‐ somally derived 30‐100 nm membranous vesicles released from many cell types into the extracellular milieu; surprisingly’ exosomes are virtually unstudied in neuro‐oncology. These microvesicles were used as vaccines in other tumor settings’ but their immunological significance is unevaluated in brain tumors. Our purpose here is to report the initial biochemical’ proteomic’ and immunological studies on murine brain tumor exosomes’ following known procedures to isolate exosomes. Our findings show that these vesicles have biophysical characteristics and proteomic profiles similar to exosomes from other cell types but that brain tumor exosomes have unique features (e.g.’ very basic isoelectric points’ expressing the mutated tumor antigen EGFRVIII and the putatively immunosuppressive cytokine TGF‐β). Administration of such exosomes into syngeneic animals produced both humoral and cellular immune responses in immunized hosts capable of rejecting subsequent tumor challenges but failed to prolong survival in established orthotopic models. Control animals received saline or cell lysate vaccines and showed no antitumor responses. Exosomes and microvesicles isolated from sera of patients with brain tumors also possess EGFR’ EGFRVIII’ and TGF‐β. We conclude that exosomes released from brain tumor cells are biochemically/biophysically like other exosomes and have immune‐modulating properties. They can escape the blood‐brain barrier’ with potential systemic and distal signaling and immune consequences.— Graner M. W. Alzate’ O. Dechkovskaia A. M. Keene J. D. Sampson J. H. Mitchell D. A. Bigner D. D. Proteomic and immunologic analyses of brain tumor exosomes. FASEBJ. 23 1541–1557 (2009)

Reduced Th17 Response in Patients with Tuberculosis Correlates with IL-6R Expression on CD4+ T Cells

RATIONALEnAlthough it is well recognized that CD4(+) T cells and T helper (Th) 1 cytokines are critical in the cell-mediated response to Mycobacterium tuberculosis, it is also clear that this immunity alone is not enough. Understanding the roles of other T cell subsets and cytokines is essential for vaccine design and clinical immunotherapy against tuberculosis (TB).nnnOBJECTIVESnTo investigate the clinical significance and possible regulatory mechanism of Th17 responses in human TB.nnnMETHODSnThe frequencies of IFN-gamma-, IL-4-, IL-17-, FoxP3- and IL-6 receptor (IL-6R)-expressing CD4(+) T cells in blood and/or pleural effusion samples of healthy donors, subjects with latent TB infection, and patients with active TB were analyzed by flow cytometry. Cytokines, transforming growth factor-beta and IL-6, in plasma and pleural fluid samples were determined by ELISA.nnnMEASUREMENTS AND MAIN RESULTSnThe frequency of Th17 cells in patients with active TB is significantly lower than those in healthy donors and individuals with latent TB infection. Correlation analysis showed that reduced Th17 responses observed in patients with active TB was significantly correlated with the decreased expression of IL-6R on CD4(+) T cells, but did not correlate with the concentrations of the cytokines, transforming growth factor-beta and IL-6. Consistently; in vitro study showed that M. tuberculosis products inhibit the expression of IL-6R on CD4(+) T cells.nnnCONCLUSIONSnOur results demonstrate that reduced Th17 responses were associated with the clinical outcome of M. tuberculosis infection. Suppression of Th17 response through down-regulation of IL-6R expression may be an important mechanism in the development of active TB.

Exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules

Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer‐associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2) or normoxic (21% O2) conditions, and exosomes secreted under hypoxic (ExoHypoxic) and normoxic (ExoNormoxic) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that ExoHypoxic have smaller average size as compared to ExoNormoxic. Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in ExoHypoxic compared to ExoNormoxic. Co‐culturing with ExoHypoxic increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. ExoHypoxic also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α‐SMA (a CAF biomarker) expression in PrSC. Compared to ExoNormoxic, ExoHypoxic showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF‐β2, TNF1α, IL6, TSG101, Akt, ILK1, and β‐catenin). Furthermore, proteome analysis revealed a higher number of proteins in ExoHypoxic (160 proteins) compared to ExoNormoxic (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, ExoHypoxic targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that ExoHypoxic are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness.

Tumor-derived chaperone-rich cell lysates are effective therapeutic vaccines against a variety of cancers

Abstract. With the clinical use of purified, tumor-derived chaperone proteins as anti-cancer vaccines already in clinical trial stages, we have focused our attention on the utility of chaperone-rich cell lysates (CRCL) in cancer immunotherapy. CRCL, as prepared from tumor lysates via a free solution-isoelectric focusing (FS-IEF) technique, is a high-yield vaccine enriched for numerous chaperone proteins. We have compared the efficacy of CRCL vaccines to that of individual chaperone protein vaccines in in vivo settings, including ELISPOT assays, tumor-growth assays and survival assays. In all experiments, CRCL vaccines were at least as effective, and in some settings perhaps even more effective, than either of the two most heavily studied components of CRCL, HSP70 and GRP94/gp96, in reduction in tumor growth and prolongation of survival in both prophylactic and pre-existing tumor settings against tumors of diverse origin and genetic background. Combining CRCL preparations with dendritic cells ex vivo resulted in a cellular vaccine that could eradicate pre-existing tumors in a high percentage of cases. The high yields of CRCL vaccines from small quantities of starting materials, the relative ease of its procurement and the functional data presented here suggest that CRCL vaccines are worthy of evaluation in pilot clinical trial cancer immunotherapy protocols.

Biodistribution and Delivery Efficiency of Unmodified Tumor-Derived Exosomes

The use of exosomes as a drug delivery vehicle has gained considerable interest. To establish if exosomes could be utilized effectively for drug delivery, a better understanding of their in vivo fate must be established. Through comparisons to liposomal formulations, which have been studied extensively for the last thirty years, we were able to make some comprehensive conclusions about the fate of unmodified tumor-derived exosomes in vivo. We observed a comparable rapid clearance and minimal tumor accumulation of intravenously-injected exosomes, PC:Chol liposomes, and liposomes formulated with the lipid extract of exosomes, suggesting that the unique protein and lipid composition of exosomes does not appreciably impact exosomes rate of clearance and biodistribution. This rapid clearance along with minimal tumor accumulation of unmodified exosomes limits their use as an anti-cancer drug delivery vehicle; however, when delivered intratumorally, exosomes remained associated with tumor tissue to a significantly greater extent than PC:Chol liposomes. Furthermore, experiments utilizing mice with impaired adaptive or innate immune systems, revealed the significance of the innate immune system along with the complement protein C5 on exosomes rate of clearance.

The heat shock response and chaperones/heat shock proteins in brain tumors: surface expression, release, and possible immune consequences.

The heat shock response is a highly conserved “stress response” mechanism used by cells to protect themselves from potentially damaging insults. It often involves the upregulated expression of chaperone and heat shock proteins (HSPs) to prevent damage and aggregation at the proteome level. Like most cancers, brain tumor cells often overexpress chaperones/HSPs, probably because of the stressful atmosphere in which tumors reside, but also because of the benefits of HSP cytoprotection. However, the cellular dynamics and localization of HSPs in either stressed or unstressed conditions has not been studied extensively in brain tumor cells. We have examined the changes in HSP expression and in cell surface/extracellular localization of selected brain tumor cell lines under heat shock or normal environments. We herein report that brain tumor cell lines have considerable heat shock responses or already high constitutive HSP levels; that those cells express various HSPs, chaperones, and at least one cochaperone on their cell surfaces; and that HSPs may be released into the extracellular environment, possibly as exosome vesicular content. In studies with a murine astrocytoma cell line, heat shock dramatically reduces tumorigenicity, possibly by an immune mechanism. Additional evidence indicative of an HSP-driven immune response comes from immunization studies using tumor-derived chaperone protein vaccines, which lead to antigen-specific immune responses and reduced tumor burden in treated mice. The heat shock response and HSPs in brain tumor cells may represent an area of vulnerability in our attempts to treat these recalcitrant and deadly tumors.

Tumor-derived multiple chaperone enrichment by free-solution isoelectric focusing yields potent antitumor vaccines.

Abstract We have utilized a free-solution/isoelectric focusing technique (FS-IEF) to obtain fractions rich in multiple chaperone proteins from clarified A20 tumor lysates. Vaccines prepared from chaperone-rich fractions are capable of providing protective immunity in mice subsequently challenged intravenously with the same A20 B cell leukemia cells. This protection is at least equal to that provided by purified, tumor-derived heat-shock protein 70, which was the best chaperone immunogen in our hands against this aggressive murine leukemia model. Dosage escalation studies, however, revealed that increasing vaccine dosages actually abrogated the protective effects. The physical nature of the enriched chaperones indicates that they are associated in complexes, which may have implications for their function. FS-IEF is relatively simple, rapid, and efficient, thus making combined multi-chaperone therapy feasible.

Medulloblastoma exosome proteomics yield functional roles for extracellular vesicles.

Medulloblastomas are the most prevalent malignant pediatric brain tumors. Survival for these patients has remained largely the same for approximately 20 years, and our therapies for these cancers cause significant health, cognitive, behavioral and developmental sequelae for those who survive the tumor and their treatments. We obviously need a better understanding of the biology of these tumors, particularly with regard to their migratory/invasive behaviors, their proliferative propensity, and their abilities to deflect immune responses. Exosomes, virus-sized membrane vesicles released extracellularly from cells after formation in, and transit thru, the endosomal pathway, may play roles in medulloblastoma pathogenesis but are as yet unstudied in this disease. Here we characterized exosomes from a medulloblastoma cell line with biochemical and proteomic analyses, and included characterization of patient serum exosomes. Further scrutiny of the proteomic data suggested functional properties of the exosomes that are relevant to medulloblastoma tumor biology, including their roles as proliferation stimulants, their activities as attractants for tumor cell migration, and their immune modulatory impacts on lymphocytes. Aspects of this held true for exosomes from other medulloblastoma cell lines as well. Additionally, pathway analyses suggested a possible role for the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A); however, inhibition of the protein’s activity actually increased D283MED cell proliferation/clonogenecity, suggesting that HNF4A may act as a tumor suppressor in this cell line. Our work demonstrates that relevant functional properties of exosomes may be derived from appropriate proteomic analyses, which translate into mechanisms of tumor pathophysiology harbored in these extracellular vesicles.