Extracellular vesicles and the tumour microenvironment

In spite of significant improvements in treatments and survival rates, metastatic breast cancer remains one of the leading causes of cancer-related deaths in women worldwide. Taxanes are microtubule-stabilizing agents largely employed as cytotoxic agents for breast cancer treatment, both in the adjuvant and neoadjuvant setting. Increasing evidence suggests that the cancer cells of primary tumors release extracellular vesicles (tEVs), which can facilitate the homing of metastatic cells to target organs. We found that paclitaxel – a lead taxane – enhances tEV release from murine breast cancer cells, both in vitro and in vivo. I will present the functional implications of this phenomenon in the context of breast cancer. Furthermore, I will discuss the determinants of microRNA sorting to EVs, and the significance of EV-mediated microRNA transfer to acceptor cells.

Apoptosis is a well-established anti-oncogenic cell-death programme. Paradoxically, high apoptosis indices in tumours are closely coupled with poor prognosis. Indeed, where it has been studied, cell loss is a striking feature of high-grade cancers, illustrating the importance of considering malignant disease as an imbalance between cell gain and cell loss rather than as a unidirectional disorder of cell gain alone. In addition to orchestrating cell loss, apoptosis can signal regenerative responses; for example compensatory proliferation in other cells. Accumulating evidence suggests that normal tissue repair and regenerative processes are hijacked in the malignant tissue microenvironment such that cancer may be likened to a “wound that fails to stop repairing”. 

We have proposed that a critical requirement for the successful growth, progression and re-growth of malignant tumours is a complex niche, conceptually termed the “onco-regenerative niche”, which is composed, in addition to transformed neoplastic cells, of normal cells and factors activated as if in tissue repair and regeneration. Our work is based around the hypothesis that tumour cell apoptosis, macrophage activation and endothelial activation are key, interlinked elements of the onco-regenerative niche and that apoptotic tumour cell-derived extracellular vesicles (Apo-EVs) provide critical intercellular communication vehicles of the niche. In aggressive B-cell lymphoma, tumour cell apoptosis promotes both angiogenesis and the accumulation of pro-tumour macrophages in the lymphoma microenvironment. Furthermore, lymphoma-derived apo-EVs have potent pro-tumour potential. These findings have important implications for the roles of apoptosis in regulation of malignant diseases and for the efficacy of apoptosis-inducing anti-cancer therapies.

Contributory talk given by Associate Professor Deborah Goberdhan. 

Cancer cells can adapt to nutrient starvation and growth inhibitory drugs by communicating with their neighbours via secreted soluble factors, exosomes and other extracellular vesicles. Exosomes are commonly believed to originate in late endosomal multivesicular bodies, but the intracellular mechanisms modulating their functions remain poorly characterised. Here a new type of exosome biogenesis pathway involving Rab11-positive recycling endosomes is identified that releases exosomes with enhanced reprogramming activities. This pathway is present in both human cancer cells and an in vivo Drosophila model of exosome secretion. Using the fly model the biogenesis of these different exosome subtypes can be visualised in living tissue for the first time. 

Inhibiting the glutamine-sensing PAT4 amino acid transporter blocks a specific form of mTORC1 signalling, leading to the preferential release of exosomes from recycling endosomes. Functionally, these stress-induced exosomes orchestrate signalling in recipient cells, which promotes pro-tumorigenic microenvironmental changes and cancer growth. Therefore in cancer cells the mTORC1 signalling hub senses microenvironmental stresses and then modulates exosome secretory pathways to drive tumour adaptation. These findings may partly explain why mTORC1 inhibitors often have poor clinical efficacy, and highlight the importance of distinguishing exosomal sub-types when defining their effects on the tumour microenvironment.

Contributory talk given by James Edgar

Edgar et al demonstrate that exosome release can be induced in HeLa cells upon inhibition of the V-ATPase, and that exosomes are observed in clusters at the plasma membrane. Such clustering shows striking similarities to clusters of retroviruses, which are attached to the plasma membrane by tetherin. To determine whether tetherin also attaches exosomes, it was knocked out and this resulted in a 4-fold reduction in plasma membrane-associated exosomes, with a concomitant increase in exosomes discharged into the medium. Such phenotype could be rescued by expression of wild-type tetherin but not tetherin lacking its GPI anchor. Edgar et al propose that tetherin may play a key role in exosome fate, determining whether they participate in long-range or short-range interactions.

Glioblastomas (GBMs, WHO grade IV astrocytomas) are the worst of the central nervous system tumors; despite maximum (and damaging) therapeutic intervention, median survival time for patients is <15 months, and overall quality of life is poor. These abysmal outcomes have changed little in 20 years. Clearly, our current therapies are inadequate; we need innovative strides in understanding GBM biology to rectify this situation. One “hot” research area is that of the impact of tumor extracellular vesicles (EVs) on normal recipient cells. Tumor EVs have extraordinary abilities to manipulate tumor microenvironments and recipient cells both proximally and distally. Tumor EVs prepare the “metastatic niche” for circulating tumor cells prior to colonization of a target organ, deflect immune responses, and alter normal cells. Thus, tumor EVs impact recipient cells to support tumor growth and progression, which undoubtedly holds true for GBMs as well. However, little is known about effects of GBM EVs on normal astrocytes—do GBM EVs drive astrocyte phenotypic changes, potentially making the astrocytes into tumor promotors? The answers could re-shape our paradigms on gliomagenesis, particularly for recurrent tumors. Here we show that GBM EVs activate cancer-type signaling pathways in recipient astrocytes, promoting astrocyte migration towards the EVs, as well as astrocyte anchorage-independent growth in soft agar. Astrocytes release of various factors to generate a tumor-promoting milieu with increased tumor cell growth. We discuss the consequences of these phenomena in the context of our current therapies with a view towards therapeutic improvement.

Cells secrete into their environment different types of extracellular vesicles (EVs) that have distinct structural and biochemical properties depending on their intracellular site of origin. Exosomes are a subtype of EVs with a mean diameter lower than 150 nm that are formed inside multivesicular compartments of the endocytic pathway. Exosomes secreted by dendritic cells (DCs) have been shown to bear functional MHC class I and class II molecules able to activate cognate T cells. These findings motivated the use of DC-derived exosomes in cancer clinical trials, although with limited clinical effects. Other EVs also bear functional immune molecules and may thus represent alternative immunotherapy tools. However, side-by-side comparison of the immune effects of exosomes and other EVs has never been comprehensively performed. Here, we have isolated different subtypes of EVs simultaneously released by live human primary DCs and extensively characterize their proteome as well as their effects on primary lymphocytes. Subtypes of EVs included large ones (> 200nm diameter) budding from the plasma membrane of live cells, small ones (< 150 nm diameter), which include exosomes and other non-exosomal sEVs, and intermediate-size EVs, which we separated by a combination of differential ultracentrifugation, floatation into density gradients and immuno-isolation via different tetraspanins. Comparative proteomic analysis of these different EVs allowed us to propose a new cartography of EV-associated protein markers, including combinations that define endosome-derived exosomes. Functionnally, we show that all these EVs efficiently induced T lymphocyte proliferation, but that they differently skew the CD4 T helper immune response. Our results provide a refined framework for the use of EVs in immunotherapy.

Cellular senescence is an important tumour suppressive mechanism to prevent the propagation of damaged cells. It induces a permanent cell cycle arrest, where the cells are metabolically and transcriptionally active. Senescent cells undergo extensive changes in gene expression, including the expression of cell cycle inhibitors and a distinctive secretory phenotype called the SASP (senescence-associated secretory phenotype), which influences the tissue microenvironment. Senescent cells accumulate in preneoplastic lesions; however, the mechanisms regulating senescence and how senescent cells alter their surrounding microenvironment is not well known. In fact, conflicting data suggests that the SASP has both anti-oncogenic and pro-oncogenic properties depending on the tissue and on the context. Here, we will explore a potential role for extracellular vesicles (EV) secreted by senescent cells in contributing to this biphasic role for the SASP during senescence activation. Our data would help identify appropriate therapeutic targets and biomarkers to tackle senescent cells and cancer at the very early stages of the disease.

Our studies elucidate the role of vesicular communication in tumor development, and how vesicular transport mechanisms can be exploited for specific targeting of treatment resistant cells of the hypoxic tumor microenvironment. Extracellular vesicles (EVs) represent relatively new signaling organelles in cell-cell communication that can carry complex biological information and elicit a pleiotropic response in recipient cells. We have found that EVs play an important role during hypoxia-mediated tumor development, and that EVs may provide a novel source of non-invasive biomarkers of the hypoxic tumor stress response. Further studies identified a key role of heparan sulfate proteoglycan-mediated endocytosis for the uptake and functional effect of EVs. More recently, we found that hypoxia down-regulates constitutive endocytosis through caveolin-1-mediated inhibition of dynamin-dependent, membrane raft endocytosis. However, several proteins can override caveolin-1 negative regulation, exhibiting increased internalization at hypoxia, further allowing antibody-mediated cytotoxin delivery and killing specifically of hypoxic cells. These data indicate that caveolin-1 modulates cell-surface proteome turnover at hypoxia with potential implications for specific targeting of the hypoxic tumor microenvironment. Based on these findings we will further explore the hypothesis that transport vesicles promote tumor aggressiveness and constitute novel therapeutic targets specifically related to typical features of the tumor microenvironment.