Co-Isolation of Cytokines and Exosomes: Implications for Immunomodulation Studies.

Exosomes play a vital role in intercellular communication and their immunomodulatory potential have become an important focus in cancer research. Various methods have been developed for the isolation although each method differs in the number and purity of exosomes they yield. In melanoma, tumor-derived exosomes drive immunosuppression within the tumor microenvironment. The co-elution of exosomes and soluble factors such as cytokines during isolation, however, make it difficult to ascertain the contribution of exosome cargo, as soluble cytokines are equally capable of immune suppression. In this review we will expound upon the biological relevance that exosome-associated cytokines possess. Furthermore, we discuss the technical challenges that arise during exosome isolation and what this means for further studies into the TME and in vivo work.

A Rapid Exosome Isolation Using Ultrafiltration and Size Exclusion Chromatography (REIUS) Method for Exosome Isolation from Melanoma Cell Lines.

Cells release extracellular vesicles (EVs) that can be detected both in vivo and in cell culture medium. Among EVs, exosomes are 50-150 nm vesicles that are systematically packaged into multivesicular bodies for release into the external environment. In cancer, these intentionally packaged exosomes carry a payload of proteins such as RNAs and surface receptors that facilitate the reprogramming of proximal cells to assemble a protumor microenvironment. Exosomes have been implicated as an important intermediary extracellular communication pathway between cells, including in melanoma. Human melanoma-derived exosomes (HMEX) have been demonstrated to modulate the extracellular environment and inhibit immune cell activation. There are many methods to isolate and enrich for exosomes and the method applied can impact yield and purity of the isolates. In this chapter we describe the REIUS (rapid exosome isolation using ultrafiltration and size exclusion chromatography) method to isolate HMEX from melanoma cell cultures and then demonstrate their enrichment using molecular and microscopic approaches.

Phase I Clinical Trial of Combination Propranolol and Pembrolizumab in Locally Advanced and Metastatic Melanoma: Safety, Tolerability, and Preliminary Evidence of Antitumor Activity.

PURPOSE: Increased ß-adrenergic receptor (ß-AR) signaling has been shown to promote the creation of an immunosuppressive tumor microenvironment (TME). Preclinical studies have shown that abrogation of this signaling pathway, particularly ß2-AR, provides a more favorable TME that enhances the activity of anti-PD-1 checkpoint inhibitors. We hypothesize that blocking stress-related immunosuppressive pathways would improve tumor response to immune checkpoint inhibitors in patients. Here, we report the results of dose escalation of a nonselective ß-blocker (propranolol) with pembrolizumab in patients with metastatic melanoma. PATIENTS AND METHODS: A 3 + 3 dose escalation study for propranolol twice a day with pembrolizumab (200 mg every 3 weeks) was completed. The primary objective was to determine the recommended phase II dose (RP2D). Additional objectives included safety, antitumor activity, and biomarker analyses. Responders were defined as patients with complete or partial response per immune-modified RECIST at 6 months. RESULTS: Nine patients with metastatic melanoma received increasing doses of propranolol in cohorts of 10, 20, and 30 mg twice a day. No dose-limiting toxicities were observed. Most common treatment-related adverse events (TRAEs) were rash, fatigue, and vitiligo, observed in 44% patients. One patient developed two grade ≥3 TRAEs. Objective response rate was 78%. While no significant changes in treatment-associated biomarkers were observed, an increase in IFNγ and a decrease in IL6 was noted in responders. CONCLUSIONS: Combination of propranolol with pembrolizumab in treatment-naïve metastatic melanoma is safe and shows very promising activity. Propranolol 30 mg twice a day was selected as RP2D in addition to pembrolizumab based on safety, tolerability, and preliminary antitumor activity.

An Immunosuppressive Effect of Melanoma-derived Exosomes on NY-ESO-1 Antigen-specific Human CD8+ T Cells is Dependent on IL-10 and Independent of BRAFV600E Mutation in Melanoma Cell Lines.

Exosomes, including human melanoma-derived exosomes (HMEX), are known to suppress the function of immune effector cells, which for HMEX has been associated with the surface presence of the immune checkpoint ligand PD-L1. This study investigated the relationship between the BRAF mutational status of melanoma cells and the inhibition of secreted HMEX exosomes on antigen-specific human T cells. Exosomes were isolated from two melanoma cell lines, 2183-Her4 and 888-mel, which are genetically wild-type BRAFWT and BRAFV600E, respectively. HMEX were isolated using a modified, size-exclusion chromatography (SEC) method shown to reduce co-isolation of non-exosome-associated cytokines compared to ultracentrifugation isolation. The immunoinhibitory effect of the exosomes was tested in vitro on patient-derived NY-ESO-1-specific CD8+ T cells challenged with NY-ESO-1 antigen. HMEX from both cell lines inhibited the immune response of antigen-specific T cells comparably, as evidenced by the reduction of IFN-γ and TNF-α in NY-ESO-1 tetramer-positive cells. This inhibition could be partially reversed by the presence of anti-PD-L1 and anti-IL-10 antibodies. IL-10 has been demonstrated to be a critical pathway for sustaining enhanced tumorigenesis in BRAFV600E mutant cells compared to BRAFWT melanoma cells. Thus, we demonstrate that HMEX inhibit antigen-specific T cell responses independent of the BRAF mutational status of the parent cells. In addition, PD-L1 and IL-10 contribute to the HMEX-mediated immunosuppression of antigen-specific human T cells. The inhibitory capacity of exosomes should be taken into consideration when developing therapies that are reliant upon the potency of customized, antigen-specific effector T cells.

Purity and yield of melanoma exosomes are dependent on isolation method.

Both exosomes and soluble factors have been implicated in the generation of an immunosuppressive tumour microenvironment. Determining the contribution of each requires stringent control of purity of the isolated analytes. The present study compares several conventional exosome isolation methods for the presence of co-enriched soluble factors while isolating exosomes from human melanoma-derived cell lines. The resultant preparations were analysed by multiplex bead array analysis for cytokine profiles, and by electron microscopy and nanotracking analysis for exosome size distribution and concentration. It is demonstrated that the amount and repertoire of soluble factors in exosome preparations is dependent upon the isolation method used. A combination of ultrafiltration and size exclusion chromatography yielded up to 58-fold more exosomes than ultracentrifugation, up to 836-fold lower concentrations of co-purified soluble factors when adjusted for exosome yield, and a greater than two-fold increase in PD-L1 expressing exosomes. Mechanistically, in context of the immunomodulatory effects of exosomes, the exosome isolation method should be carefully considered in order to limit any effects due instead to co-eluted soluble factors.

A Review of Exosomes and their Role in The Tumor Microenvironment and Host-Tumor "Macroenvironment".

Tumor-derived exosomes (TEX) are important intercellular messengers that contribute to tumorigenesis and metastasis through a variety of mechanisms such as immunosuppression and metabolic reprogramming that generate a pre-metastatic niche favorable to tumor progression. Our lab has contributed further to the understanding of the miRNA payloads in TEX by demonstrating that human melanoma-derived exosome (HMEX) associated miRNAs contribute to the metabolic reprogramming of normal stroma. This mini-review highlights the role of TEX in the tumor microenvironment (TME) and the hypothesis that exosomes may also generate a host-tumor "macroenvironment" beyond the TME through their miRNA and protein payloads, so to speak "fertilizing the soil for cancer seeding."

Publisher Correction: Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Low-Dose Ketamine for Postoperative Pain Management.

Ketamine, an anesthetic agent, is gaining attention as an analgesic for the management of acute and chronic pain conditions. Perianesthesia nurses may expect to see ketamine's use increase as more anecdotal and evidence-based experience is gained with its use for pain management. Unlike opioids, ketamine supports respirations while supporting hemodynamic function; moreover, the agent has potential for decreasing opioid-induced hyperalgesia. Ongoing clinical evidence continues to support ketamine's use for analgesia, thus it may be argued that the current Food and Drug Administration classification for ketamine as an anesthetic agent is outdated, and patients would be better served by a reclassification of this medication to include its use for analgesic purposes. This continuing education article provides an overview of ketamine, its side effects, and the possible adverse reactions so perianesthesia nurses may be prepared to care for postsurgical patients who receive ketamine for analgesic purposes.

Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment.

Local acidification of stroma is proposed to favour pre-metastatic niche formation but the mechanism of initiation is unclear. We investigated whether Human Melanoma-derived exosomes (HMEX) could reprogram human adult dermal fibroblasts (HADF) and cause extracellular acidification. HMEX were isolated from supernatants of six melanoma cell lines (3 BRAF V600E mutant cell lines and 3 BRAF wild-type cell lines) using ultracentrifugation or Size Exclusion Chromatography (SEC). Rapid uptake of exosomes by HADF was demonstrated following 18 hours co-incubation. Exposure of HDAF to HMEX leads to an increase in aerobic glycolysis and decrease in oxidative phosphorylation (OXPHOS) in HADF, consequently increasing extracellular acidification. Using a novel immuno-biochip, exosomal miR-155 and miR-210 were detected in HMEX. These miRNAs were present in HMEX from all six melanoma cell lines and were instrumental in promoting glycolysis and inhibiting OXPHOS in tumour cells. Inhibition of miR-155 and miR-210 activity by transfection of miRNA inhibitors into HMEX reversed the exosome-induced metabolic reprogramming of HADF. The data indicate that melanoma-derived exosomes modulate stromal cell metabolism and may contribute to the creation of a pre-metastatic niche that promotes the development of metastasis.

Ketamine Infusions for Outpatient Pain Management: A Policy Development Project.

Current literature supports using ketamine for both acute and chronic pain management. It is imperative that the development of evidence-based protocols and policies keep pace with health care delivery to ensure patient safety. This project's objective was to formulate an outpatient ketamine infusion policy that promotes consistent and evidence-based care within a specified hospital system. This policy addresses potential side effects and minimization of adverse events by addressing patient selection, level of nursing care required, appropriate monitoring, and staff education.

Novel tropolones induce the unfolded protein response pathway and apoptosis in multiple myeloma cells.

Tropolones are small organic compounds with metal-directing moieties. Tropolones inhibit the proliferation of cancer cell lines, possibly through their effects on metalloenzymes such as select histone deacetylases (HDACs). Pan-HDAC inhibitors are therapeutically beneficial in the treatment of multiple myeloma, however there is interest in the use of more selective HDAC inhibitor therapy to minimize adverse side effects. We hypothesized that tropolones might have anti-myeloma activities. To this end, a series of novel α-substituted tropolones were evaluated for effects on multiple myeloma cells. While all tested tropolones showed some level of cytotoxicity, MO-OH-Nap had consistently low IC50 values between 1-11 µM in all three cell lines tested and was used for subsequent experiments. MO-OH-Nap was found to induce apoptosis in a concentration-dependent manner. Time course experiments demonstrated that MO-OH-Nap promotes caspase cleavage in a time frame that was distinct from the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Furthermore, MO-OH-Nap- and SAHA-treated cells possess unique gene expression patterns, suggesting they promote apoptosis via different mechanisms. In particular, MO-OH-Nap increases the expression of markers associated with endoplasmic reticulum stress and the unfolded protein response. Synergistic cytotoxic effects were observed when cells were treated with the combination of MO-OH-Nap and the proteasome inhibitor bortezomib. However, treatment with MO-OH-Nap did not abrogate the bortezomib-induced increase in aggresomes, consistent with an HDAC6-independent mechanism for the observed synergy. Collectively, these finding support further investigation into the usefulness of α-substituted tropolones as anti-myeloma agents.

Ketamine for Pain Management-Side Effects & Potential Adverse Events.

An old anesthetic agent, ketamine is finding new use in lower doses for analgesic purposes. There are concerns stemming from its potential side effects-specifically psychomimetic effects. These side effects are directly related to dose amount. The doses used for analgesic purposes are much lower than those used for anesthesia purposes. A literature review was performed to ascertain potential side effects and/or adverse events when using ketamine for analgesia purposes. The search included CINAHL, PubMed, and Ovid using the search terms "ketamine," "ketamine infusion," "pain," "adverse events," "practice guideline," and "randomized controlled trial." Searches were limited to full-text, peer-reviewed articles and systematic reviews. Initially 1,068 articles were retrieved. The search was then narrowed by using the Boolean connector AND with various search term combinations. After adjusting for duplication, article titles and abstracts were reviewed, leaving 25 articles for an in-depth analysis. Specific exclusion criteria were then applied. The literature supports the use of ketamine for analgesic purposes, and ketamine offers a nonopioid option for the management of some pain conditions. Because ketamine is still classified as an anesthetic agent, health care institutions should develop their own set of policies and protocols for the administration of ketamine. By using forethought and understanding of the properties of ketamine, appropriate care may be planned to mitigate potential side effects and adverse events so that patients are appropriately cared for and their pain effectively managed.

Bishomoisoprenoid triazole bisphosphonates as inhibitors of geranylgeranyl diphosphate synthase.

Protein geranylgeranylation reactions are dependent on the availability of geranylgeranyl diphosphate (GGDP), which serves as the isoprenoid donor. Inhibition of GGDP synthase (GGDPS) is of interest from a drug development perspective as GGDPS inhibition results in impaired protein geranylgeranylation, which in multiple myeloma, disrupts monoclonal protein trafficking and induces apoptosis. We have recently reported a series of isoprenoid triazole bisphosphonates and have demonstrated that a 3:1 mixture of homogeranyl and homoneryl isomers potently, and in a synergistic manner, inhibits GGDPS. We now present the synthesis and biological evaluation of a novel series of bishomoisoprenoid triazoles which furthers our understanding of the structure-function relationship of this class. These studies demonstrate the importance of chain length and olefin stereochemistry on inhibitory activity.

Olefin Isomers of a Triazole Bisphosphonate Synergistically Inhibit Geranylgeranyl Diphosphate Synthase.

The isoprenoid donor for protein geranylgeranylation reactions, geranylgeranyl diphosphate (GGDP), is the product of the enzyme GGDP synthase (GGDPS) that condenses farnesyl diphosphate (FDP) and isopentenyl pyrophosphate. GGDPS inhibition is of interest from a therapeutic perspective for multiple myeloma because we have shown that targeting Rab GTPase geranylgeranylation impairs monoclonal protein trafficking, leading to endoplasmic reticulum stress and apoptosis. We reported a series of triazole bisphosphonate GGDPS inhibitors, of which the most potent was a 3:1 mixture of homogeranyl (HG) and homoneryl (HN) isomers. Here we determined the activity of the individual olefin isomers. Enzymatic and cellular assays revealed that although HN is approximately threefold more potent than HG, HN is not more potent than the original mixture. Studies in which cells were treated with varying concentrations of each isomer alone and in different combinations revealed that the two isomers potentiate the induced-inhibition of protein geranylgeranylation when used in a 3:1 HG:HN combination. A synergistic interaction was observed between the two isomers in the GGDPS enzyme assay. These results suggested that the two isomers bind simultaneously to the enzyme but within different domains. Computational modeling studies revealed that HN is preferred at the FDP site, that HG is preferred at the GGDP site, and that both isomers may bind to the enzyme simultaneously. These studies are the first to report a set of olefin isomers that synergistically inhibit GGDPS, thus establishing a new paradigm for the future development of GGDPS inhibitors.

A new motif for inhibitors of geranylgeranyl diphosphate synthase.

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure-function relationship which is dependent on the nature of the alkyl group at the α-carbon.

Stereocontrolled regeneration of olefins from epoxides.

Through treatment with NaI and trifluoroacetic anhydride, which presumably forms trifluoroacetyl iodide in situ, epoxides can be converted to olefins. This reaction now has been shown to tolerate remote olefins without loss of their individual stereochemistry. A reaction sequence involving regiospecific epoxidation of an isoprenoid alcohol, conversion of the alcohol to an azide, and cycloaddition with an acetylene, followed by conversion of the epoxide back to the original olefin, has allowed stereocontrolled preparation of triazole bisphosphonates with a farnesyl or a geranylgeranyl substituent. This strategy may be applicable selective protection of an alkene in other polyolefins, including substrates for metathesis reactions.

Potent Triazole Bisphosphonate Inhibitor of Geranylgeranyl Diphosphate Synthase.

Studies of triazole bisphosphonates have resulted in identification of a potent inhibitor of geranylgeranyl diphosphate synthase (IC50 = 45 nM) with very good selectivity for this enzyme over farnesyl diphosphate synthase (IC50 = 28 µM). This compound also potently disrupts geranylgeranylation and induces cytotoxicity in human myeloma cells at submicromolar levels, suggesting that it may serve as a lead compound for treatment of malignancies characterized by excessive protein secretion.

Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells.

Multiple myeloma (MM) is characterized by the production of monoclonal protein (MP). We have shown previously that disruption of the isoprenoid biosynthetic pathway (IBP) causes a block in MP secretion through a disruption of Rab GTPase activity, leading to an enhanced unfolded protein response and subsequent apoptosis in MM cells. Autophagy is induced by cellular stressors including nutrient deprivation and ER stress. IBP inhibitors have been shown to have disparate effects on autophagy. Here we define the mechanisms underlying the differential effects of IBP inhibitors on autophagic flux in MM cells utilizing specific pharmacological inhibitors. We demonstrate that IBP inhibition induces a net increase in autophagy as a consequence of disruption of isoprenoid biosynthesis which is not recapitulated by direct geranylgeranyl transferase inhibition. IBP inhibitor-induced autophagy is a cellular defense mechanism as treatment with the autophagy inhibitor bafilomycin A1 enhances the cytotoxic effects of GGPP depletion, but not geranylgeranyl transferase inhibition. Immunofluorescence microscopy studies revealed that IBP inhibitors disrupt ER to Golgi trafficking of monoclonal light chain protein and that this protein is not a substrate for alternative degradative pathways such as aggresomes and autophagosomes. These studies support further development of specific GGTase II inhibitors as anti-myeloma agents.

N-Oxide derivatives of 3-(3-pyridyl)-2-phosphonopropanoic acids as potential inhibitors of Rab geranylgeranylation.

The N-oxide derivatives of [2-(3-pyridinyl)-1-hydroxyethylidene-1,1-phosphonocarboxylic acid (or PEHPC) and [2-(3-pyridinyl)-1-ethylidene-1,1-phosphonocarboxylic acid (or PEPC) have been prepared and evaluated for their activity against several enzymes which utilize isoprenoids. The parent pyridines are known inhibitors of GGTase II, but the N-oxide derivatives show no improvement in biological activity in assays with the isolated enzyme. However, the PEHPC N-oxide did induce significant accumulation of intracellular light chain in myeloma cells, consistent with inhibition of Rab geranylgeranylation.

Packed red cell transfusions alter mesenteric arterial reactivity and nitric oxide pathway in preterm lambs.

BACKGROUND: Cases of necrotizing enterocolitis occurring within 48 h of packed red blood cell (PRBC) transfusions are increasingly being described in observational studies. Transfusion-associated gut injury is speculated to result from an abnormal mesenteric vascular response to transfusion. However, the mechanism of disruption of the balance between mesenteric vasoconstriction and relaxation following transfusion is not known. METHODS: Preterm lambs (n = 16, 134 d gestation; term: 145-147 d) were delivered and ventilated for 24 h. All the lambs received orogastric feeds with colostrum. In addition, 10 of these lambs received PRBC transfusions. Vasoreactivity was evaluated in isolated mesenteric arterial rings using norepinephrine and endothelin-1 as vasoconstrictors. Endothelium-dependent (A23187, a calcium ionophore) and endothelium-independent (SNAP) nitric oxide (NO) donors were used as vasorelaxants. Mesenteric arterial endothelial NO synthase (eNOS), soluble guanylyl cyclase (sGC), and phosphodiesterase 5 (PDE5) mRNA analyses and protein assays were performed. RESULTS: Transfusion with PRBC significantly increased mesenteric vasoconstriction to norepinephrine and endothelin-1 and impaired relaxation to A23187 and SNAP. Mesenteric arterial eNOS protein decreased following PRBC transfusion. No significant changes were noted in sGC and PDE5 mRNA or protein assays. CONCLUSION: PRBC transfusion in enterally fed preterm lambs promotes mesenteric vasoconstriction and impairs vasorelaxation by reducing mesenteric arterial eNOS.