Kidney Cancer and Potential Use of Urinary Extracellular Vesicles.

Kidney cancer is the 14th most common cancer globally. The 5-year relative survival rate of kidney cancer at a localized stage is 92.9% and it declines to 17.4% in metastatic stage. Currently, the most accurate method of its diagnosis is tissue biopsy. However, the invasive and costly nature of biopsies makes it undesirable in many patients. Therefore, novel biomarkers for diagnosis and prognosis should be explored. Urinary extracellular vesicles (uEVs) are small vesicles (50-200 nm) in urine carrying nucleic acids, proteins and lipids as their cargos. These uEVs' cargos can provide non-invasive alternative to monitor kidney health. In this review, we have summarized recent studies investigating potential use of uEVs' cargos as biomarkers in kidney cancer for diagnosis, prognosis and therapeutic intervention.

Milk-borne small extracellular vesicles: kinetics and mechanisms of transport, distribution, and elimination.

Small extracellular vesicles (sEVs) in milk have the qualities desired for delivering therapeutics to diseased tissues. The production of bovine milk sEVs is scalable (1021 annually per cow), and they resist degradation in the gastrointestinal tract. Most cells studied to date internalize milk sEVs by a saturable process that follows Michaelis-Menten kinetics. The bioavailability of oral milk sEVs is approximately 50%. In addition to crossing the intestinal mucosa, milk sEVs also cross barriers such as the placenta and blood-brain barrier, thereby enabling the delivery of therapeutics to hard-to-reach tissues. In time course studies, levels of milk sEVs peaked in the intestinal mucosa, plasma, and urine approximately 6 h and returned to baseline 24 h after oral gavage in mice. In tissues, milk sEV levels peaked 12 h after gavage. Milk sEVs appear to be biologically safe. No cytokine storm was observed when milk sEVs were added to cultures of human peripheral blood mononuclear cells or administered orally to rats. Liver and kidney function and erythropoiesis were not impaired when milk sEVs were administered to rats by oral gavage for up to 15 days. Protocols for loading milk sEVs with therapeutic cargo are available. Currently, the use of milk sEVs (and other nanoparticles) in the delivery of therapeutics is limited by their rapid elimination through internalization by macrophages and lysosomal degradation in target cells. This mini review discusses the current knowledge base of sEV tissue distribution, excretion in feces and urine, internalization by macrophages, and degradation in lysosomes.

The RNA cargo in small extracellular vesicles from chicken eggs is bioactive in C57BL/6 J mice and human peripheral blood mononuclear cells ex vivo.

Small extracellular vesicles (sEVs) and their RNA cargo in milk are bioavailable in humans, pigs, and mice, and their dietary depletion and supplementation elicits phenotypes. Little is known about the content and biological activity of sEVs in foods of animal origin other than milk. Here we tested the hypothesis that sEVs in chicken eggs (Gallus gallus) facilitate the transfer of RNA cargo from an avian species to humans and mice, and their dietary depletion elicits phenotypes. sEVs were purified from raw egg yolk by ultracentrifugation and authenticated by transmission electron microscopy, nano-tracking device, and immunoblots. The miRNA profile was assessed by RNA-sequencing. Bioavailability of these miRNAs in humans was assessed by egg feeding study in adults, and by culturing human peripheral blood mononuclear cells (PBMCs) with fluorophore-labeled egg sEVs ex vivo. To further assess bioavailability, fluorophore-labeled miRNAs, encapsulated in egg sEVs, were administered to C57BL/6 J mice by oral gavage. Phenotypes of sEV RNA cargo depletion were assessed by feeding egg sEV and RNA-defined diets to mice and using spatial learning and memory in the Barnes and water mazes as experimental readouts. Egg yolk contained 6.30 × 1010 ± 6.06 × 109 sEVs/mL, which harbored eighty-three distinct miRNAs. Human PBMCs internalized sEVs and their RNA cargo. Egg sEVs, loaded with fluorophore-labeled RNA and administered orally to mice, accumulated primarily in brain, intestine and lungs. Spatial learning and memory (SLM) was compromised in mice fed on egg sEV- and RNA-depleted diet compared to controls. Egg consumption elicited an increase of miRNAs in human plasma. We conclude that egg sEVs and their RNA cargo probably are bioavailable. The human study is registered as a clinical trial and accessible at https://www.isrctn.com/ISRCTN77867213.

Review: Milk Small Extracellular Vesicles for Use in the Delivery of Therapeutics.

Small extracellular vesicles (sEVs, "exosomes") in milk have attracted considerable attention for use in delivering therapeutics to diseased tissues because of the following qualities. The production of milk sEVs is scalable, e.g., more than 1021 sEVs may be obtained annually from a single cow. Milk EVs protect their cargo against degradation in the gastrointestinal tract and during industrial processing. Milk sEVs and their cargo are absorbed following oral administration and they cross barriers such as intestinal mucosa, placenta and the blood-brain barrier in humans, pigs, and mice. Milk sEVs do no alter variables of liver and kidney function, or hematology, and do not elicit immune responses in humans, rats, and mice. Protocols are available for loading milk sEVs with therapeutic cargo, and a cell line is available for assessing effects of milk sEV modifications on drug delivery. Future research will need to assess and optimize sEV shelf-life and storage and effects of milk sEV modifications on the delivery of therapeutic cargo to diseased tissues.

Therapeutic miRNA-Enriched Extracellular Vesicles: Current Approaches and Future Prospects.

Extracellular vesicles (EVs) are 50-300 nm vesicles secreted by eukaryotic cells. They can carry cargo (including miRNA) from the donor cell to the recipient cell. miRNAs in EVs can change the translational profile of the recipient cell and modulate cellular morphology. This endogenous mechanism has attracted the attention of the drug-delivery community in the last few years. EVs can be enriched with exogenous therapeutic miRNAs and used for treatment of diseases by targeting pathological recipient cells. However, there are some obstacles that need to be addressed before introducing therapeutic miRNA-enriched EVs in clinics. Here, we focused on the progress in the field of therapeutic miRNA enriched EVs, highlighted important areas where research is needed, and discussed the potential to use them as therapeutic miRNA carriers in the future.

Downregulation of glypican-4 facilitates breast cancer progression by inducing cell migration and proliferation.

Glypican-4 (GPC-4) is a heparan sulphate glycoprotein, associated with cell membrane via a Glycosyl phosphatidyl (GPI)-anchor. It is involved in cell migration, cell growth, differentiation and morphogenesis as well as chemoresistance and cancer stem cell maintenance in pancreatic cancer. However, its role in breast cancer remains unclear. To elucidate the role of GPC-4 in breast cancer, we analyzed GPC-4 expression in breast cancer patients and breast cancer cell lines. Our results demonstrated that GPC-4 expression was downregulated in metastatic tumors as compared to non-metastatic tumors. Further, GPC4's downregulation was confirmed in breast cancer metastatic cells (MDA-MBA-231 and MDA-MB-LM2) compared to non-metastatic cells (T47-D and MCF-7) with quantitative PCR and western blot. Knock-down of GPC-4 in non-metastatic cells significantly increased cell-migration and invasion. Similarly, over-expressing GPC-4 in metastatic cells decreased cell-migration/invasion and cell proliferation. Additionally, GPC-4 overexpression decreased in-vivo tumorigenicity in nude mice. Therefore, this research for the first time, has established the role of glypican-4 as a tumor-suppressor in breast cancer by decreasing migration and proliferation, revealing it as a possible therapy for breast cancer.

Exosomes in Food: Health Benefits and Clinical Relevance in Diseases.

Exosomes are membrane-bound organelles generally secreted by eukaryotic cells that contain mRNAs, microRNAs, and/or proteins. However, recent studies have reported the isolation of these particles from foods such as lemon, ginger, and milk. Owing to their absorption by intestinal cells and further travel via the bloodstream, exosomes can reach distant organs and affect overall health in both infants and adults. The potential role of food-derived exosomes (FDEs) in alleviating diseases, as well as in modulating the gut microbiota has been shown, but the underlying mechanism is still unknown. Moreover, exosomes may provide biocompatible vehicles for the delivery of anti-cancer drugs, such as doxorubicin. Thus, exosomes may allow medical nutritionists and clinicians to develop safe and targeted therapies for the treatment of various pathologies. The present review introduces FDEs and their contents, highlights their role in disease and infant/adult health, and explores their potential use as therapeutic agents.

Promotion of tumor progression and cancer stemness by MUC15 in thyroid cancer via the GPCR/ERK and integrin-FAK signaling pathways.

Thyroid cancer is the fifth most common cancer diagnosed in women worldwide. Notwithstanding advancements in the prognosis and treatment of thyroid cancer, 10-20% of thyroid cancer patients develops chemotherapeutic resistance and experience relapse. According to previous reports and TCGA database, MUC15 (MUCIN 15) upregulation is highly correlated with thyroid cancer progression. However, the role of MUC15 in tumor progression and metastasis is unclear. This study aimed to investigate factors mediating cancer stemness in thyroid cancer. MUC15 plays an important role in sphere formation, as an evident from the expression of stemness markers including SOX2, KLF4, ALDH1A3, and IL6. Furthermore, ectopic expression of MUC15 activated extracellular signal-regulated kinase (ERK) signaling via G-protein-coupled receptor (GPCR)/cyclic AMP (cAMP) and integrin/focal adhesion kinase pathways. Interestingly, ectopic expression of MUC15 did not affect RAF/mitogen-activated protein kinase kinase (MEK)-mediated ERK activation. The present findings may provide novel insights into the development of diagnostic, prognostic, and therapeutic applications of MUC15 in thyroid cancer.

mirRICH, a simple method to enrich the small RNA fraction from over-dried RNA pellets.

Techniques to isolate the small RNA fraction (<200nt) by column-based methods are commercially available. However, their use is limited because of the relatively high cost. We found that large RNA molecules, including mRNAs and rRNAs, are aggregated together in the presence of salts when RNA pellets are over-dried. Moreover, once RNA pellets are over-dried, large RNA molecules are barely soluble again during the elution process, whereas small RNA molecules (<100nt) can be eluted. We therefore modified the acid guanidinium thiocyanate-phenol-chloroform (AGPC)-based RNA extraction protocol by skipping the 70% ethanol washing step and over-drying the RNA pellet for 1 hour at room temperature. We named this novel small RNA isolation method "mirRICH." The quality of the small RNA sequences was validated by electrophoresis, next-generation sequencing, and quantitative PCR, and the findings support that our newly developed column-free method can successfully and efficiently isolate small RNAs from over-dried RNA pellets.