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.

No abstract

Patient and rodent solid tumors often exhibit elevated interstitial fluid pressure (IFP). This condition is recognized as a prognostic indicator for reduced responses to therapy and decreased disease-free survival. Here we tested whether induction of a thermoregulatory-mediated rise in tissue blood flow, induced by exposure of mice to mild environmental heat stress, could influence IFP and other vascular parameters within tumors. Using several murine tumor models, we found that heating results in a sustained reduction in tumor IFP correlating with increased tumor vascular perfusion (measured by fluorescent imaging of perfused vessels, laser Doppler and magnetic resonance imaging) as well as a sustained reduction in tumor hypoxia. When radiation therapy was administered 24 hours post-heating, we also observed a significant improvement in efficacy that may be a result of the sustained reduction in tumor hypoxia. These data suggest, for the first time, that environmental manipulation of normal vasomotor function is capable of achieving therapeutically beneficial changes in IFP and microvascular function in the tumor microenvironment.

The self assembly of phospholipid molecules in the bilayer form was considered in terms of equivalent molecular shapes representing intermolecular forces. The equivalent size of each phospholipid headgroup was approximated by the net atomic volume plus the volume of the associated water molecules, which was derived from water/ hydrocarbon partitioning experiments. The equivalent lengths ofunsaturated acyl chains were derived from the retention time data from chromatographic measurements. The spontaneous curvature of various phospholipid monolayers was calculated from their equivalent molecular shapes, and the energy required to flatten them to the bilayer plane was calculated, using the known bending modulus. With increasing bending energy, the mixtures showed increasing susceptibility to phospholipase A2, facilitated lipid tansfer rate by phospholipid exchange proteins, permeability to carboxyfluorescein, incorporation of human erythrocyte proteins, and calcium transport by CaATPase from sarcoplasmic reticulum in reconstituted vesicles. When the calculation was applied to known lipid compositions of nine cellular membranes, the protein/lipid ratio and phospholipid/cholesterol ratio were found to have a positive and a negative correlation, respectively, with the latent bending energy of the phospholipids. The energy expense in conforming to a bilayer phase may be an important physical parameter regarding the activity and the biogenesis of membranes.The regulated diversity in lipid composition of biological membranes is well recognized, but the need for such diversity is not fully understood. Apparently, the lipids in biological membranes do more than provide a passive barrier and a supporting matrix for membrane proteins. There is ample evidence that changes in lipid structure can influence the activity ofmembranes (1). The quest for maintaining a certain "fluidity" fails to explain the diversity of membrane lipid species. It is known that not all lipids found in biological membranes form bilayers when dispersed in water. In fact, a large portion of the membrane lipids do not form bilayers at physiological conditions (pH, calcium concentration, and temperature). These so-called "nonbilayer" lipids generally prefer high curvature structures such as inverted hexagonal (HI,), inverted cubic, and "lipid particles" when dispersed in water (1, 2). However, no such structures have ever been found in functioning biomembranes. It would thus seem that the ability of these lipid mixtures to form high curvature structures upon self assembly, rather than the inverted structures themselves, is needed for the proper functioning of biological membranes.The polymorphism or phase preference of any lipid is governed by the coherence force, which is the combined result of the exclusion volume, polarity, headgroup interactions, and van der Waals interactions between hydrocarbon chains, among others (3, 4). These forces are difficult to account for analytically; therefore the quantitative assessment of the energetics of bilay...

It has been shown that there is a correlation between the fusogenecity of synthetic peptides corresponding to the N-terminal segment of wild-type and mutant forms of simian immunodeficiency virus gp32 (SIV) and their mode of insertion into lipid bilayers. Fusogenic activity is only observed when the peptide inserts into the bilayer with an oblique orientation. Since bilayer destabilization is a necessary step in membrane fusion, we investigate how fusion peptides, which insert at different orientations into lipid bilayers, structurally affect model membranes. We use X-ray diffraction to investigate the structural effects of two synthetic peptides on three different lipid systems. One peptide corresponds to the wild-type sequence (SIVwt), which inserts into the membrane at an oblique angle and is fusogenic. The other peptide has a rearranged sequence (SIVmutV), inserts into the membrane along the bilayer normal, and is nonfusogenic. Our results are expressed through different structural effects, which depend on the lipid system: for example, (i) disordering of the L alpha phase as evidenced by the broadening of the diffraction peaks, (ii) morphological convertion of multilamellar vesicles into unilamellar vesicles, (iii) decrease of the hexagonal phase cell parameter when SIVwt is added, and (iv) change in the conditions for the formation of cubic phases as well as its kinetic stability over a range of temperatures. Some of these observations are explicable based on the fact that the SIVwt destabilizes bilayers by inducing a negative monolayer curvature, while the SIVmutV destabilizes bilayers by inducing a positive monolayer curvature. Finally, we present a model which describes how these findings correlate with fusogenic activity and fusion inhibitory activity, respectively.