Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers.

BackgroundThe need to accurately discriminate dendritic cells (DCs) and macrophages (Mphs) in mouse lungs is critical given important biological differences. However, a validated flow cytometry–based method is still lacking, resulting in much confusion between both cell types.MethodsSingle‐cell suspensions freshly obtained from collagenase‐digested lung tissue were stained with a CD11c‐specific monoclonal antibody, detected using a PE‐Cy5 or APC‐conjugated secondary reagent. Cellular immunophenotype was simultaneously explored using a panel of PE‐conjugated markers. The FL1 or FITC‐detection channel was reserved for the assessment of autofluorescence.ResultsCD11c‐bright cells were heterogeneous and displayed a bimodal distribution with regard to autofluorescence (AF). CD11c+/low‐AF cells were lineage‐negative and showed features compatible with myeloid DCs. This was confirmed by morphology, potent T‐cell stimulatory function in a mixed‐leukocyte reaction, surface expression of MHCII and costimulatory molecules, and further immunophenotypical criteria, including the expression of Mac‐1 and absence of CD8α. In contrast, CD11c+/high‐AF cells displayed the features of pulmonary Mphs, including typical Mph morphology, very weak induction of T‐cell proliferation, low to absent expression of MHCII and costimulatory molecules, and very low levels of Mac‐1 as well as F4/80. We also show that only CD11c+/high‐AF cells strongly expressed the macrophage marker MOMA‐2, while interestingly Mac‐3 was expressed at high levels by CD11c+/high‐AF and low‐AF alike.ConclusionsThis study shows that the combination of CD11c‐expression and autofluorescence is necessary and sufficient to accurately separate DCs from macrophage subpopulations in mouse lungs. © 2004 Wiley‐Liss, Inc.

Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. It is caused by mutations in the inflammasome adaptor Pyrin, but how FMF mutations alter signaling in FMF patients is unknown. Herein, we establish Clostridium difficile and its enterotoxin A (TcdA) as Pyrin-activating agents and show that wild-type and FMF Pyrin are differentially controlled by microtubules. Diverse microtubule assembly inhibitors prevented Pyrin-mediated caspase-1 activation and secretion of IL-1β and IL-18 from mouse macrophages and human peripheral blood mononuclear cells (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon microtubule disassembly in PBMCs of FMF patients but not in cells of patients afflicted with other autoinflammatory diseases. We further demonstrate that microtubules control Pyrin activation downstream of Pyrin dephosphorylation and that FMF mutations enable microtubule-independent assembly of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) micrometer-sized perinuclear structures (specks). The discovery that Pyrin mutations remove the obligatory requirement for microtubules in inflammasome activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations.

More than many other fields in medicine, cancer vaccine development has been plagued by a wide gap between the massive amounts of highly encouraging preclinical data on one hand, and the disappointing clinical results on the other. It is clear now that traditional approaches from the infectious diseases' vaccine field cannot be borrowed as such to treat cancer. This review highlights some of the strategies developed to improve vaccine formulations for oncology, including research into more powerful or “smarter” adjuvants to elicit anti-tumoral cellular immune responses. As an illustration of the difficulties in translating smart preclinical strategies into real benefit for the cancer patient, the difficult road of vaccine development in lung cancer is given as example. Finally, an outline is provided of the combinatorial strategies that leverage the increasing knowledge on tumor-associated immune suppressive networks. Indeed, combining with drugs that target the dominant immunosuppressive pathway in a given tumor promises to unlock the true power of cancer vaccines and potentially offer long-term protection from disease relapse.