Spheresomes are the main extracellular vesicles in low-grade gliomas.

Cancer progression and its impact on treatment response and prognosis is deeply regulated by tumour microenvironment (TME). Cancer cells are in constant communication and modulate TME through several mechanisms, including transfer of tumour-promoting cargos through extracellular vesicles (EVs) or oncogenic signal detection by primary cilia. Spheresomes are a specific EV that arise from rough endoplasmic reticulum-Golgi vesicles. They accumulate beneath cell membrane and are released to the extracellular medium through multivesicular spheres. This study describes spheresomes in low-grade gliomas using electron microscopy. We found that spheresomes are more frequent than exosomes in these tumours and can cross the blood-brain barrier. Moreover, the distinct biogenesis processes of these EVs result in unique cargo profiles, suggesting different functional roles. We also identified primary cilia in these tumours. These findings collectively contribute to our understanding of glioma progression and metastasis.

Liver tissue remodeling following ablation with irreversible electroporation in a porcine model.

Irreversible electroporation (IRE) is a method of non-thermal focal tissue ablation characterized by irreversibly permeabilizing the cell membranes while preserving the extracellular matrix. This study aimed to investigate tissue remodeling after IRE in a porcine model, especially focusing on the extracellular matrix and hepatic stellate cells. IRE ablation was performed on 11 female pigs at 2,000 V/cm electric field strength using a versatile high-voltage generator and 3 cm diameter parallel-plate electrodes. The treated lobes were removed during surgery at 1, 3, 7, 14, and 21 days after IRE. Tissue remodeling and regeneration were assessed by histopathology and immunohistochemistry. Throughout the treated area, IRE led to extensive necrosis with intact collagenous structures evident until day 1. From then on, the necrosis progressively diminished while reparative tissue gradually increased. During this process, the reticulin framework and the septal fibrillar collagen remained in the necrotic foci until they were invaded by the reparative tissue. The reparative tissue was characterized by a massive proliferation of myofibroblast-like cells accompanied by a complete disorganization of the extracellular matrix with the disappearance of hepatic architecture. Hepatic stellate cell markers were associated with the proliferation of myofibroblast-like cells and the reorganization of the extracellular matrix. Between 2 and 3 weeks after IRE, the lobular architecture was almost completely regenerated. The events described in the present study show that IRE may be a valid model to study the mechanisms underlying liver regeneration after extensive acute injury.

Primary cilia presence and implications in bladder cancer progression and invasiveness.

Urothelial bladder cancer is the tenth most common cancer worldwide. It is divided into muscle and non-muscle invading bladder cancer. Primary cilia have been related to several cancer hallmarks such as proliferation, epithelial-to-mesenchymal transition (EMT) or tumoral progression mainly through signaling pathways as Hedgehog (Hh). In the present study, we used immunohistochemical and ultrastructural techniques in human tissues of healthy bladder, non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) to study and clarify the activation of epithelial-to-mesenchymal transition and Hedgehog signaling pathway and the presence of primary cilia. Thus, we found a clear correlation between EMT and Hedgehog activation and bladder cancer stage and progression. Moreover, we identified the presence of primary cilia in these tissues. Interestingly, we found that in NMIBC, some ciliated cells cross the basement membrane and localized in lamina propria, near blood vessels. These results show a correlation between EMT beginning from urothelial basal cells and primary cilia assembly and suggest a potential implication of this structure in tumoral migration and invasiveness (likely in a Hh-dependent way). Hence, primary cilia may play a fundamental role in urothelial bladder cancer progression and suppose a potential therapeutic target.

Hedgehog signalling pathway activation in gastrointestinal stromal tumours is mediated by primary cilia.

BACKGROUND: Gastrointestinal stromal tumour (GIST) is a mesenchymal cancer which derives from interstitial cells of Cajal. To determine whether a relationship between Hedgehog (Hh) signalling pathway and primary cilia exists in GIST tumours is intended here. METHODS: Immunohistochemical, immunofluorescence and ultrastructural techniques were performed in this study. RESULTS: We show that GIST cells present primary cilia (an antenna-like structure based on microtubules). But, moreover, we prove Hedgehog signalling pathway activation in these tumours (a pathway related with tumoural features such as proliferation, migration or stemness) and we show for the first time that this signalling pathway activation in GIST is mediated by primary cilia, likely in a paracrine way. CONCLUSION: Thus, primary cilia and Hedgehog signalling would be fundamental in tumoural microenvironment control of GIST cells for their maintenance, differentiation and proliferation.

Ultrastructural and immunohistochemical study of phenotypic switch in gastrointestinal smooth muscle cells.

Dedifferentiation is a loss of phenotypic specialization that converts differentiated cells into adult stem cells in order to proliferate and differentiate into replacement tissue. This occurs in several tissues from various organs, such as smooth muscle cells (SMCs) of the mammalian gastrointestinal tract. The aim of this study was to describe ultrastructural and immunohistochemical changes in SMCs which could be compatible with a dedifferentiation process in human and rabbit intestinal muscles. Ultrastructural study and immunohistochemical staining (SMemb and MyoD) on human and rabbit duodenum tissue sections were performed. In both species, this dedifferentiation process is characterized by a loss of intercellular junctions, increased intercellular spaces, cytoskeletal disorganization, perinuclear accumulation of large vacuoles that tend to fuse, rupture of the vacuole membrane and release of cytoplasmic fragments. Dedifferentiated cells show the characteristic phenotype of a mesenchymal cell with scarce perinuclear cytoplasm, long cytoplasmic prolongations and finely distributed granular chromatin in the nucleus. These morphological changes are accompanied by a modulation to a less mature phenotype showing immunoreactivity for the embryonic form of the myosin heavy chain and for the myogenic regulatory factor MyoD. We suggest that SMC dedifferentiation includes the elimination of the contractile apparatus, the activation of the nucleus and the re-expression of embryonic markers. We described an ultrastructural dedifferentiation process possible in intestinal SMCs. This dedifferentiation process seems to play a key role in the homeostasis of the intestinal muscle.

Cell damage produced by magnetic fluid hyperthermia on microglial BV2 cells.

We present evidence on the effects of exogenous heating by water bath (WB) and magnetic hyperthermia (MHT) on a glial micro-tumor phantom. To this, magnetic nanoparticles (MNPs) of 30-40 nm were designed to obtain particle sizes for maximum heating efficiency. The specific power absorption (SPA) values (f = 560 kHz, H = 23.9 kA/m) for as prepared colloids (533-605 W/g) dropped to 98-279 W/g in culture medium. The analysis of the intracellular MNPs distribution showed vesicle-trapped MNPs agglomerates spread along the cytoplasm, as well as large (~0.5-0.9 µm) clusters attached to the cell membrane. Immediately after WB and MHT (T = 46 °C for 30 min) the cell viability was ≈70% and, after 4.5 h, decreased to 20-25%, demonstrating that metabolic processes are involved in cell killing. The analysis of the cell structures after MHT revealed a significant damage of the cell membrane that is correlated to the location of MNPs clusters, while local cell damage were less noticeable after WB without MNPs. In spite of the similar thermal effects of WB and MHT on the cell viability, our results suggest that there is an additional mechanism of cell damage related to the presence of MNPs at the intracellular space.

Magnetic Nanoparticles for Efficient Delivery of Growth Factors: Stimulation of Peripheral Nerve Regeneration.

The only clinically approved alternative to autografts for treating large peripheral nerve injuries is the use of synthetic nerve guidance conduits (NGCs), which provide physical guidance to the regenerating stump and limit scar tissue infiltration at the injury site. Several lines of evidence suggest that a potential future strategy is to combine NGCs with cellular or molecular therapies to deliver growth factors that sustain the regeneration process. However, growth factors are expensive and have a very short half-life; thus, the combination approach has not been successful. In the present paper, we proposed the immobilization of growth factors (GFs) on magnetic nanoparticles (MNPs) for the time- and space-controlled release of GFs inside the NGC. We tested the particles in a rat model of a peripheral nerve lesion. Our results revealed that the injection of a cocktail of MNPs functionalized with nerve growth factor (NGF) and with vascular endothelial growth factor (VEGF) strongly accelerate the regeneration process and the recovery of motor function compared to that obtained using the free factors. Additionally, we found that injecting MNPs in the NGC is safe and does not impair the regeneration process, and the MNPs remain in the conduit for weeks.

Biogenesis of a new type of extracellular vesicles in gastrointestinal stromal tumors: ultrastructural profiles of spheresomes.

Extracellular vesicles (EVs) have emerged as an intercellular communication mediator in cancer. They seem to be involved in tumor processes by means of transformation of surrounding cells previous to metastasis by transferring miRNAs and oncogenic proteins. It is known that EVs, depending on their source, can be exosomes or ectosomes. Although the first type constitutes a specific population formed from the endosomal system, via multivesicular bodies, the ectosome biogenesis is not yet well known. In this study, we report a new type of EVs which has been termed spheresomes. While exosomes come from multivesicular bodies and ectosomes from direct budding of plasma membrane, spheresomes present a new mechanism of shedding from a spherical membrane structure which we have named multivesicular spheres. These EVs are first described in gastrointestinal stromal tumor cells in the present study. But moreover, these new membrane spherical structures appear not only next to tumoral cells but also different distances from them. Since some other authors have evidenced oncogenic KIT-containing EVs, it is also suggested here that surrounding cells uptake of these first described EVs, GIST-derived spheresomes, could induce tumor invasiveness. That is why the prevention of signaling processes developed by these new EVs may represent an alternative approach for GIST treatment.

Primary cilia in gastric gastrointestinal stromal tumours (GISTs): an ultrastructural study.

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal (non-epithelial) neoplasms of the human gastrointestinal (GI) tract. They are thought to derive from interstitial cells of Cajal (ICCs) or an ICC progenitor based on immunophenotypical and ultrastructural similarities. Because ICCs show primary cilium, our hypothesis is based on the possibility that some of these neoplastic cells could also present it. To determine this, an exhaustive ultrastructural study has been developed on four gastric GISTs. Previous studies had demonstrated considerable variability in tumour cells with two dominating phenotypes, spindly and epithelioid. In addition to these two types, we have found another cell type reminiscent of adult ICCs with a voluminous nucleus surrounded by narrow perinuclear cytoplasm with long slender cytoplasmic processes. We have also noted the presence of small undifferentiated cells. In this study, we report for the first time the presence of primary cilia (PCs) in spindle and epithelioid tumour cells, an ultrastructural feature we consider of special interest that has hitherto been ignored in the literature dealing with the ultrastructure of GISTs. We also point out the frequent occurrence of multivesicular bodies (MVBs). The ultrastructural findings described in gastric GISTs in this study appear to be relevant considering the critical roles played by PCs and MVBs recently demonstrated in tumourigenic processes.