Role of microenvironmental acidity and tumor exosomes in cancer immunomodulation.

Tumor microenvironment (TME) is a complex milieu in which tumor grows, develops and progresses through a complex bi-directional cross-talk with immune-, stromal cells, and the extracellular matrix (ECM). In this context, tumor-derived exosomes (TE) drive the fate of tumor cells through a stimulatory or inhibitory role on immune system. In fact, TE can induce the apoptosis of cells of the immune surveillance, and enhance the proliferation and survival of stromal cells that sustain tumor development. However, depending on the molecular cargo, TE are also able to stimulate anti-tumor immune response. TME is mainly characterized by the acidic pH that contributes to tumor development, through multiple mechanisms. Among these, the impairment of tumor immune surveillance does occur within acidic TME, and is directly mediated by acidic pH or by molecular cargo carried by TE. Little is known about the role of TE in immunomodulation in acidic conditions. The present review summarizes the studies describing the role of microenvironmental acidity and TE in immune system modulation.

IL-33 Promotes CD11b/CD18-Mediated Adhesion of Eosinophils to Cancer Cells and Synapse-Polarized Degranulation Leading to Tumor Cell Killing.

Eosinophils are major effectors of Th2-related pathologies, frequently found infiltrating several human cancers. We recently showed that eosinophils play an essential role in anti-tumor responses mediated by immunotherapy with the 'alarmin' intereukin-33 (IL-33) in melanoma mouse models. Here, we analyzed the mechanisms by which IL-33 mediates tumor infiltration and antitumor activities of eosinophils. We show that IL-33 recruits eosinophils indirectly, via stimulation of tumor cell-derived chemokines, while it activates eosinophils directly, up-regulating CD69, the adhesion molecules ICAM-1 and CD11b/CD18, and the degranulation marker CD63. In co-culture experiments with four different tumor cell lines, IL-33-activated eosinophils established large numbers of stable cell conjugates with target tumor cells, with the polarization of eosinophil effector proteins (ECP, EPX, and granzyme-B) and CD11b/CD18 to immune synapses, resulting in efficient contact-dependent degranulation and tumor cell killing. In tumor-bearing mice, IL-33 induced substantial accumulation of degranulating eosinophils within tumor necrotic areas, indicating cytotoxic activity in vivo. Blocking of CD11b/CD18 signaling significantly reduced IL-33-activated eosinophils' binding and subsequent killing of tumor cells, indicating a crucial role for this integrin in triggering degranulation. Our findings provide novel mechanistic insights for eosinophil-mediated anti-tumoral function driven by IL-33. Treatments enabling tumor infiltration and proper activation of eosinophils may improve therapeutic response in cancer patients.

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules.

BACKGROUND: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. METHODS: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. RESULTS: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. CONCLUSIONS: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.

Cell Propagation of Cholera Toxin CTA ADP-Ribosylating Factor by Exosome Mediated Transfer.

In this study, we report how the cholera toxin (CT) A subunit (CTA), the enzyme moiety responsible for signaling alteration in host cells, enters the exosomal pathway, secretes extracellularly, transmits itself to a cell population. The first evidence for long-term transmission of CT's toxic effect via extracellular vesicles was obtained in Chinese hamster ovary (CHO) cells. To follow the CT intracellular route towards exosome secretion, we used a novel strategy for generating metabolically-labeled fluorescent exosomes that can be counted by flow cytometry assay (FACS) and characterized. Our results clearly show the association of CT with exosomes, together with the heat shock protein 90 (HSP90) and Protein Disulfide Isomerase (PDI) molecules, proteins required for translocation of CTA across the ER membrane into the cytoplasm. Confocal microscopy showed direct internalization of CT containing fluorescent exo into CHO cells coupled with morphological changes in the recipient cells that are characteristic of CT action. Moreover, Me665 cells treated with CT-containing exosomes showed an increase in Adenosine 3',5'-Cyclic Monophosphate (cAMP) level, reaching levels comparable to those seen in cells exposed directly to CT. Our results prompt the idea that CT can exploit an exosome-mediated cell communication pathway to extend its pathophysiological action beyond an initial host cell, into a multitude of cells. This finding could have implications for cholera disease pathogenesis and epidemiology.

Combining Type I Interferons and 5-Aza-2'-Deoxycitidine to Improve Anti-Tumor Response against Melanoma.

Resistance to IFN-I-induced antineoplastic effects has been reported in many tumors and arises, in part, from epigenetic silencing of IFN-stimulated genes by DNA methylation. We hypothesized that restoration of IFN-stimulated genes by co-administration of the demethylating drug 5-aza-2'-deoxycitidine (decitabine [DAC]) may enhance the susceptibility to IFN-I-mediated antitumoral effects in melanoma. We show that combined administration of IFN-I and DAC significantly inhibits the growth of murine and human melanoma cells, both in vitro and in vivo. Compared with controls, DAC/IFN-I-treated melanoma cells exhibited reduced cell growth, augmented apoptosis, and diminished migration. Moreover, IFN-I and DAC synergized to suppress the growth of three-dimensional human melanoma spheroids, altering tumor architecture. These direct antitumor effects correlated with induction of the IFN-stimulated gene Mx1. In vivo, DAC/IFN-I significantly reduced melanoma growth via stimulation of adaptive immunity, promoting tumor-infiltrating CD8+ T cells while inhibiting the homing of immunosuppressive CD11b+ myeloid cells and regulatory T cells. Accordingly, exposure of human melanoma cells to DAC/IFN-I induced the recruitment of immune cells toward the tumor in a Matrigel (Corning Life Sciences, Kennebunkport, ME)-based microfluidic device. Our findings underscore a beneficial effect of DAC plus IFN-I combined treatment against melanoma through both direct and immune-mediated anti-tumor effects.

Generation, Quantification, and Tracing of Metabolically Labeled Fluorescent Exosomes.

Over the last 10 years, the constant progression in exosome (Exo)-related studies highlighted the importance of these cell-derived nano-sized vesicles in cell biology and pathophysiology. Functional studies on Exo uptake and intracellular trafficking require accurate quantification to assess sufficient and/or necessary Exo particles quantum able to elicit measurable effects on target cells. We used commercially available BODIPY(®) fatty acid analogues to label a primary melanoma cell line (Me501) that highly and spontaneously secrete nanovesicles. Upon addition to cell culture, BODIPY fatty acids are rapidly incorporated into major phospholipid classes ultimately producing fluorescent Exo as direct result of biogenesis. Our metabolic labeling protocol produced bright fluorescent Exo that can be examined and quantified with conventional non-customized flow cytometry (FC) instruments by exploiting their fluorescent emission rather than light-scattering detection. Furthermore, our methodology permits the measurement of single Exo-associated fluorescence transfer to cells making quantitative the correlation between Exo uptake and activation of cellular processes. Thus the protocol presented here appears as an appropriate tool to who wants to investigate mechanisms of Exo functions in that it allows for direct and rapid characterization and quantification of fluorescent Exo number, intensity, size, and eventually evaluation of their kinetic of uptake/secretion in target cells.

Cell-to-cell propagation of the bacterial toxin CNF1 via extracellular vesicles: potential impact on the therapeutic use of the toxin.

Eukaryotic cells secrete extracellular vesicles (EVs), either constitutively or in a regulated manner, which represent an important mode of intercellular communication. EVs serve as vehicles for transfer between cells of membrane and cytosolic proteins, lipids and RNA. Furthermore, certain bacterial protein toxins, or possibly their derived messages, can be transferred cell to cell via EVs. We have herein demonstrated that eukaryotic EVs represent an additional route of cell-to-cell propagation for the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1). Our results prove that EVs from CNF1 pre-infected epithelial cells can induce cytoskeleton changes, Rac1 and NF-κB activation comparable to that triggered by CNF1. The observation that the toxin is detectable inside EVs derived from CNF1-intoxicated cells strongly supports the hypothesis that extracellular vesicles can offer to the toxin a novel route to travel from cell to cell. Since anthrax and tetanus toxins have also been reported to engage in the same process, we can hypothesize that EVs represent a common mechanism exploited by bacterial toxins to enhance their pathogenicity.

Modification of proteins secreted by endothelial cells during modeled low gravity exposure.

The exposure of the human body to microgravity, conditions that occurs during space flights, causes significant changes in the cardiovascular system. Many cell types have been involved in these changes, and the endothelium seems to play a major role. In endothelial cells (EC), it has been shown that modeled low gravity impairs nitric oxide synthesis, cell adhesion, extracellular matrix composition, cytoskeleton organization, cytokines, and growth factors secretion. Nevertheless, detailed analysis of EC physiological changes induced by microgravity exposure is still lacking. Secretome analysis is one of the most promising approaches for the identification of biomarkers directly related to the physiopathological cellular state. In this study, we analyzed in details the modifications of EC secretome by using umbilical vein endothelial (HUVE) cells exposed to modeled low gravity conditions. By adopting a two-dimensional (2-D) proteomic approach, in conjunction with a technique for the compression of the dynamic range of proteins, we observed that modeled low gravity exposure of HUVE cells affected the secretion of proteins involved in the regulation of cytoskeleton assembly. Moreover, by using Luminex® suspension array systems, we found that the low gravity condition decreased in ECs the secretion of some key pro-inflammatory cytokines, including IL-1α and IL-8, and of the pro-angiogenic factor bFGF. On the contrary, microgravity increase the secretion of two chemokines (Rantes and Eotaxin), involved in leukocytes recruitment.

[Formaldehyde in air of indoor and outdoor environments of urban area, relationships man's exposure]. / Formaldeide in aria di ambienti interni ed esterni di un'area urbana, in relazione all'esposizione dell'uomo.

This study presents the results of man's exposure to formaldehyde, carried out in indoor and outdoor environments in the city of Ferrara. The processes were obtained on average concentration measurements carried out for one month by indoor, outdoor and personal samplings. Concentration values obtained by personal samplings (PE) were compared to both outdoor and indoor values, the latter including domestic and working environments. The results showed that there was a significant difference between formaldeyde concentrations outdoor and PE (p = 0,03 < alpha = 0,05), while a significant difference was not found between indoor and PE. Indoor values (values as average of 38 samples) were 19,5 microg/m3, outdoor ones were 6,9 microg/m3 and PE 19,9 microg/m3. The average indoor value in domestic environments (20,7 microg/m3) was higher than the value found in working environments (17,9 microg/m3). Binary comparisons showed that indoor and outdoor, together with indoor and personal samplings, display the same variability of formaldehyde concentrations.