Differential fates of biomolecules delivered to target cells via extracellular vesicles.

Extracellular vesicles (EVs), specifically exosomes and microvesicles (MVs), are presumed to play key roles in cell-cell communication via transfer of biomolecules between cells. The biogenesis of these two types of EVs differs as they originate from either the endosomal (exosomes) or plasma (MVs) membranes. To elucidate the primary means through which EVs mediate intercellular communication, we characterized their ability to encapsulate and deliver different types of macromolecules from transiently transfected cells. Both EV types encapsulated reporter proteins and mRNA but only MVs transferred the reporter function to recipient cells. De novo reporter protein expression in recipient cells resulted only from plasmid DNA (pDNA) after delivery via MVs. Reporter mRNA was delivered to recipient cells by both EV types, but was rapidly degraded without being translated. MVs also mediated delivery of functional pDNA encoding Cre recombinase in vivo to tissues in transgenic Cre-lox reporter mice. Within the parameters of this study, MVs delivered functional pDNA, but not RNA, whereas exosomes from the same source did not deliver functional nucleic acids. These results have significant implications for understanding the role of EVs in cellular communication and for development of EVs as delivery tools. Moreover, studies using EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer.

[99mTc]Annexin V-128 SPECT Monitoring of Splenic and Disseminated Listeriosis in Mice: a Model of Imaging Sepsis.

PURPOSE: Here, we evaluate [(99m)Tc]annexin V-128, an in vivo marker of apoptosis, for single photon emission computed tomography (SPECT) imaging of localization and antibiotic treatment of disseminated bacterial infection, using a well-described mouse model that employs bioluminescent Listeria monocytogenes. PROCEDURES: Sixteen groups of five mice in six separate experiments were infected with bioluminescent Listeria, and in vivo bioluminescence imaging (BLI) was performed each day, to assess the location and extent of infection and response to antibiotics. [(99m)Tc]annexin V-128 was then injected for SPECT imaging, and the two sets of images were correlated and validated. RESULTS: Signals from BLI and [(99m)Tc]annexin V-128 SPECT co-localized within the spleen and other organs including bone marrow, intestine, nasopharynx, and brain. Decreases in [(99m)Tc]annexin V-128 uptake and BLI signal within the spleen directly reflected the reduction of bacterial infection by ampicillin treatment. CONCLUSIONS: Tc-99m-Annexin V-128 uptake as observed by SPECT allowed for the detection of systemic listeriosis and ampicillin treatment in mice. [(99m)Tc]annexin V-128 should be further explored for the assessment of bacterial spread and antibiotic efficacy in patients with disseminated bacterial infection.

A killer choice for cancer immunotherapy.

The promise of cell-based immunotherapies for the treatment of cancer offers the potential of therapeutic synergy with chemo- and radiotherapies that may overcome current limitations leading to durable responses and prevention of recurrence. There is a wide array of cell-based immunotherapies that are either poised to enter cancer clinical trials or are in clinical trials, and many are showing some success. Yet within this field, there are clear obstacles that need to be overcome, including limited access across tissue barriers, development of antigen tolerance, and the immunosuppressive microenvironment of tumors. Through an understanding of immune cell signaling and trafficking, immune cell populations can be selected for adoptive transfer, and delivery strategies can be developed that circumvent these obstacles to effectively direct populations of cells with robust anti-tumor efficacy to the target. Within the realm of immune cell therapies, cytokine-induced killer (CIK) cells have demonstrated promising trafficking patterns, effective delivery of synergistic therapeutics, and stand-alone efficacy. Here, we discuss the next generation of CIK therapies and their application for the effective treatment of a wide variety of cancers.

Monitoring dynamic interactions between breast cancer cells and human bone tissue in a co-culture model.

PURPOSE: Bone is a preferential site of breast cancer metastasis, and models are needed to study this process at the level of the microenvironment. We have used bioluminescence imaging (BLI) and multiplex biomarker immunoassays to monitor dynamic breast cancer cell behaviors in co-culture with human bone tissue. PROCEDURES: Femur tissue fragments harvested from hip replacement surgeries were co-cultured with luciferase-positive MDA-MB-231-fLuc cells. BLI was performed to quantify breast cell proliferation and track migration relative to bone tissue. Breast cell colonization of bone tissues was assessed with immunohistochemistry. Biomarkers in co-culture supernatants were profiled with MILLIPLEX(®) immunoassays. RESULTS: BLI demonstrated increased MDA-MB-231-fLuc cell proliferation (p < 0.001) in the presence vs. absence of bones and revealed breast cell migration toward bone. Immunohistochemistry illustrated MDA-MB-231-fLuc cell colonization of bone, and MILLIPLEX(®) profiles of culture supernatants suggested breast/bone crosstalk. CONCLUSIONS: Breast cell behaviors that facilitate metastasis occur reproducibly in human bone tissue co-cultures and can be monitored and quantified using BLI and multiplex immunoassays.