Cancer cachexia is a multifactorial metabolic syndrome characterized by the rapid loss of skeletal muscle mass with or without the loss of fat mass. Nearly 50–80% of all cancer patients' experience rapid weight loss results in ∼20% of cancer-related deaths. The levels of pro-inflammatory and pro-cachectic factors were significantly up-regulated in cachexia patients when compared with the patients who were without cachexia. It is becoming evident that these factors work synergistically to induce cancer cachexia. Extracellular vesicles (EVs) including exosomes and microvesicles are implicated in cell–cell communication, immune response, tissue repair, epigenetic regulation, and in various diseases including cancer. It has been reported that these EVs regulate cancer progression, metastasis, organotropism and chemoresistance. In recent times, the role of EVs in regulating cancer cachexia is beginning to unravel. The aim of this mini article is to review the recent knowledge gained in the field of EVs and cancer cachexia. Specifically, the role of tumour cell-derived EVs in promoting catabolism in distally located skeletal muscles and adipose tissue will be discussed.
Cachexia is a metabolic syndrome characterized by the rapid loss of skeletal muscle mass with or without the loss of fat mass . Cachexia occurs in patients with cancer, heart failure, rheumatoid arthritis, obstructive pulmonary disease, and during chronic infections [2,3]. Approximately 9 million patients globally are affected by cachexia and it is considered as a positive risk factor for death [4,5]. In the late stage of cancer, cachexia is considered to be one of the most distressing phases, which not only causes functional impairment and psychological distress but also impedes anticancer treatment . Owing to muscle wasting and weakened conditions, major surgeries in cachexic patients are considered high risk and chemotherapy is highly toxic to endure . Intrinsic properties of cancer cachexia include chronically inflamed state while extrinsic factors include the reduction in physical activity, anorexia, asthenia and fatigue that leads to poor quality of life [8,9]. Nearly 50–80% of all cancer patients experience rapid weight loss and it is estimated that cachexia accounts for 20% of cancer-related deaths . The frequency of weight loss in most of the cancers depends on various factors such as cancer type, site and mass, host genotype and associated signalling pathways . It was observed that pancreatic/gastric cancer have the highest frequency of weight loss, whereas non-Hodgkin's lymphoma, breast cancer, acute non-lymphocytic leukaemia, and sarcomas have the lowest frequency of weight loss . Although negative protein–energy balance coupled with hyper-catabolism, hypo-anabolism, and systemic inflammation results in the loss of body weight , it is unclear as how muscle wasting is differentially regulated based on the cancer site. As muscle wasting cannot be reversed by nutritional supplementation, the higher weight loss positively correlates with low survival rates . Death normally occurs through hypostatic pneumonia due to the extensive loss of respiratory muscles and cardiac failure when the patient has lost ∼30–40% of their body weight .
Mediators of cancer cachexia
Over the last few decades, there has been immense interest in understanding the regulators of cancer cachexia. Various cytokines and cachectic factors that could regulate the loss of skeletal muscle mass and lipolysis in adipose tissue have been documented. These pro-inflammatory and pro-cachectic factors produced by the tumour cells are considered to have important roles in the genesis and progression of cachexia. For instance, pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNF)-α, interleukin (IL)-1, IL-6, and anti-inflammatory cytokine such as IL-10, promote the activation of transcription factors associated with the muscle wasting and have a critical role in the pathological mechanisms involving cachexia [15–17]. These factors co-operate with each other (IL-6 and TNF-α) or act alone as drivers of systemic inflammation. The molecular mechanism by which pro-inflammatory factors contribute to the development of cancer cachexia involves the activation of various downstream transcription factors and molecules in the myotubes and adipocytes. TNF-α induces the catabolism of various myofibrillar proteins in the myotubes via activation of nuclear transcription factor-kappa B (NF-κB) through IkB kinase signalling. The activated NF-κB increases the transcription of genes such as muscle RING finger protein-1 which codes for E3 ligase—one of the enzyme in ubiquitin proteasome pathway. E3 ligases specifically degrade myofibrillar proteins such as actin, myosin heavy and light chains, and troponin-I and there by contributing to the loss of lean body mass during cancer cachexia [15,18,19]. Similarly, IL-6 induces apoptosis in skeletal muscles by increased caspase activity via activation of janus kinase/signal transducer and activator of transcription-3 and mitogen-activated protein kinase (MAPK) cascades. Along with the TNF-α and IL-6, recent preclinical study in murine models of anorexia–cachexia syndrome demonstrated that higher levels of IL-10 inhibited protein synthesis in skeletal muscles via increased levels of Myc and activation of mTOR signalling [20,21]. In adipose tissue, pro-inflammatory factors cause browning of white adipose tissue (WAT). The switch from WAT to brown adipose tissue (BAT) known as WAT browning occurs due to the increased expression of uncoupling protein 1 which uncouples mitochondrial respiration towards thermogenesis instead of ATP synthesis and there by leading to increased lipid mobilization and energy expenditure. It was observed that the process of WAT browning occurs much before than skeletal muscle atrophy [16,22].
In addition to pro-inflammatory cytokines, pro-cachectic factors such as lipid-mobilizing factor (LMF)  and proteolysis-inducing factor (PIF) are also produced by cachexia-inducing tumours. LMF is a zinc-α2-glycoprotein that can sensitize adipocytes to lipolytic stimuli, catabolizes tri-acylglycerols to fatty acids, and has a direct lipolytic effect in WAT . PIF is reported as a chief candidate for promoting skeletal muscle atrophy and acts by decreasing protein synthesis through phosphorylation of eukaryotic translation initiation factor 2α. In addition, PIF accelerates protein degradation through the ubiquitin–proteasome pathway and regulates muscle atrophy . Similarly, myostatin is another pro-cachectic factor that is secreted by the skeletal muscles, adipose tissue, and tumour cells. Myostatin acts via activin receptor type II-mediated signalling and regulates muscle wasting [25,26]. Further to the pro-inflammatory and pro-cachectic factors, cachexia is also activated by neuroendocrine stress responses via release of glucocorticoids. The targeted deletion of the glucocorticoid receptor in mice muscles exhibited 77% less skeletal muscle atrophy than control animals in response to tumour growth . Though the muscle cells and nervous system cross-talk is not fully demonstrated, it is thought that this mechanism is complex and further studies are needed to understand the role of pituitary hormones in inducing muscle wasting and lipolysis. Although the levels of pro-inflammatory and pro-cachectic factors were significantly up-regulated in cancer cachexia patients , it is becoming evident that these factors have a synergistic effect . For instance, although etanercept-based TNF-α inhibition improved cachexia-associated fatigue in a cohort of cancer patients , clinical trials with TNF-α blocking antibodies showed no benefit, suggesting that inhibition of TNF-α alone is not sufficient to treat cancer cachexia .
Extracellular vesicles in cancer cachexia
Cells secrete or shed a large variety of extracellular vesicles (EVs) into the extracellular space [32,33]. The EVs include exosomes (30–150 nm in diameter), apoptotic bodies (50–5000 nm) and ectosomes or shedding microvesicles (100–1000 nm) (Figure 1) [34–37]. Exosomes are secreted by the cells when the multivesicular bodies fuse with the plasma membrane and hence are of endocytic origin . On the contrary, ectosomes and apoptotic bodies are released by the budding of the plasma membrane from live or apoptotic cells, respectively . EVs contain nucleic acids, proteins, and various metabolites such as amino acids, lipids, and TCA cycle intermediates that are reflective of the cell type of origin and state of the cell, i.e. transformed, differentiated, stimulated, and stressed [40–45]. In recent years, much attention was given to EVs because of their participation in intercellular communication, and various physiological and pathological processes including tumour progression . The possible reason for cells choosing EVs for intercellular communication might be due to the fact that EVs provide stable conformational conditions for the nucleic acids and proteins, conserve bioactivity, improve bio-distribution, and support an efficient interaction with target cells. In the last decade, it is well established that EVs can form the pre-metastatic niche and can regulate metastatic organotropism [46–49]. It has been hypothesized that integrins present on tumour-derived exosomes could facilitate organ-specific metastatic behaviour of the cancer cells. Current reports have shown that tumour-derived EVs have a role in disease progression and could transport cargo that might profoundly influence remote tissues such as skeletal muscle and adipose tissue (Figure 2). Cancer cachexia is primarily caused due to the loss of skeletal muscles and/or WAT . Whether cachexia is largely driven by a tumour or as a result of the host response to a tumour is yet to be fully investigated. Our understanding about the role of EVs in cancer cachexia has improved and there are few interesting studies that have been published that support the role of cancer-derived EVs in muscle and fat loss.
Different subtypes of EVs.
Tumour-induced muscle atrophy, lipolysis, and browning.
Zhang et al.  demonstrated that cachectic cancer cells (Lewis lung carcinoma and C-26 mouse colon carcinoma) release EVs which could potentially induce muscle wasting. Tumour cell released heat shock proteins 70 and 90 (Hsp70/90)-associated EVs that are critical to induce muscle catabolism resulting in muscle wasting through the activation of Toll-like receptor 4 (TLR4). TLR4 activates p38-mitogen-activated protein kinase (p38-MAPK), which in turn activates both the ubiquitin proteasome pathway that degrades specific regulatory and myofibrillar proteins involved in muscle protein expression and the autophagy–lysosome pathway that degrades mitochondria and other cellular components. The role of p38-MAPK was further confirmed by the increase in the levels of atrogin 1 and UBR2, which get activated via C/EBPβ and the up-regulation of atrogin 1 and UBR2 positively correlates with the rate of catabolism of muscle. Supporting this hypothesis, increase in the levels of autophagy marker LC3 both in in vitro and in vivo models in the presence of Hsp70/90 EVs specified the contribution of tumour-released EVs in the catabolism of skeletal and adipose tissues. It was also observed that the systemic increase in circulating levels of inflammatory cytokines such as TNF-α and IL-6 during cachexia was also mainly due to the activation of TLR4 via EVs associated with Hsp70/90. These levels were abrogated upon down-regulation of Rab27 GTPase (that control the different steps of EV release) further strengthening the role of EVs in inducing systemic inflammation. Furthermore, the presence of EVs leads to the loss or reduction of myosin heavy chain in differentiated C2C12 which significantly affects the muscle strength and results in muscle atrophy. Over all, the study by Zhang et al. explained the molecular mechanism of tumour-derived EVs in inducing muscle atrophy in distally located skeletal muscles .
Sagar et al. demonstrated that pancreatic cancer (PC)-derived EVs induce lipolysis in murine and human subcutaneous adipocytes . Furthermore, the study provided considerable insights into the entry of PC-EVs and the mechanism by which these internalized EVs induce lipolysis via the activation of various signalling pathways. PC-EVs consist of adrenomedullin (AM), a 52 amino acid (∼10 kDa) peptide which is ubiquitously expressed in many cells including adipocytes. Notably, AM levels are elevated in PC-EVs isolated from patients plasma compared with EVs from non-PC control subjects. Lipolysis-inducing factor AM binds to adrenomedullin receptors (ADMRs), a member of the seven transmembrane domain G-protein-coupled receptor superfamily, on the surface of adipocyte and activates ERK1, ERK2 and p38-MAPK pathways and induces lipolysis. Upon internalization of EVs via micropinocytosis or caveolin-mediated endocytosis, EV-associated AM mediates their effect through the interaction with ADMRs and increases the expression of phosphorylated hormone-sensitive lipase (p-HSL), a marker for active lipolysis. Along with p-HSL, it also elevates the expression of phosphorylated perilipin1, which is a marker for an early event of lipolysis. ADMR blockers/inhibitors of p38 and MAPK decreased lipolysis. In addition, it was also shown that PC-EVs does not contain functionally significant TNF-α, a well-known lipolytic factor. Sagar et al.'s  report suggested that the loss of body weight in PC is due to the loss of adipocytes via the release of AM-associated EVs.
Wang et al. showed that there was substantial inhibition of adipogenesis upon internalization of A549 lung cancer-secreted EVs by human adipose tissue-derived mesenchymal stem cells (hAD-MSCs). The decrease of lipid droplets in hAD-MSCs, lower mRNA expression of the adipogenic transcription factor PPARγ and adipocyte-specific marker lipoprotein lipase upon treatment with EVs further strengthened the halt in adipogenesis. It has been suggested that the anti-adipogenic effects were due to the activation of transforming growth factor beta signalling pathway which increases the levels of phosphorylated Smad2 (pSmad2), leads to nuclear localization of Smad4 and thereby decreases the adipogenesis in hAD-MSCs .
Role of miRNAs and EV-associated miRNAs in cachexia
Recent studies have established that microRNAs (miRNAs) are pivotal in regulating the metabolic status, biological processes, and disease progression . In cancer-associated cachexia, it was shown that miR-486 decreases the expression of Forkhead box O1 (FoxO1), one of the member of E3 ubiquitin ligases, thereby decreasing the activation of the ubiquitin proteasome system and muscle protein loss. In contrast, miR-21 and miR-206 have been shown to induce cachexia . For instance, miR-21-containing EVs promote catabolism of muscles by activating TLR7 or TLR8 on the mouse and human myoblasts, respectively. The activated TLR7/8 requires c-Jun N-terminal kinase to induce the apoptosis of the muscles and thereby contributes to the muscle mass wasting or atrophy . In addition to muscle catabolism, miRNAs also regulate several aspects of adipocyte function. Global miRNA expression of abdominal subcutaneous adipose tissue from the gastric cancer patients with and without muscle loss revealed that miR-378 expression was up-regulated. Follow-up analysis revealed that miR-378 modulates key lipolytic proteins such as LIPE, PLIN1, and PNPLa2 thereby inducing catecholamine-activated lipolysis. Several miRNAs including miR-483-5p, miR-23a, miR-744 and miR-99b that are implicated in adipogenesis and mitochondrial β-oxidation were down-regulated in adipose tissue [57–59]. In view of the dual role of miRNAs in cancer-associated cachexia and inflammation, it is critical to study the biogenesis, exportation and the mechanism by which tumour-derived miRNAs induce catabolism in the distally located tissues.
It is important to consider that cancer-associated weight loss first came to light almost 100 years ago, yet there are only a few reports on understanding its trigger, mechanism, possible mediators, and biomarkers. In line with the high mortality rate in cancer patients associated with cachexia, addressing cachexia is critical to manage cancer therapy. Research on targeting cachectic factors provided us clues that controlling the levels of a single factor or mediator may not be equally effective in all cancer types and during all the three cachectic stages. It is also clear that various cancers induce muscle wasting and lipolysis at various rates and via different mechanisms. Hence, it is important to consider multiple factors while developing treatment options for cancer cachexia. Perhaps, individualize treatment options may be the best choice depending on the cancer type and the BMI of the cachectic patients. Currently, very few drugs and inhibitors are in development for treating weight loss in advanced cancers and most of them are in different phases of clinical trials. Recently, Johnston et al. targeted Fn14 (tumour necrosis factor receptor superfamily member 12A) with monoclonal antibodies and were able to attenuate cachexia in animal models. The anti-Fn14 monoclonal antibody not only restored the body mass but also significantly reduced the expression of inflammatory markers such as IL-6 and TNF-α .
Research on the role of EVs in various diseases including neurodegenerative diseases , autoimmune disorders , inflammatory diseases , musculoskeletal diseases  and cancer have advanced our understanding of initiation, establishment and progression of the diseases. Along with the biology, EVs have immense potential to be utilized for the identification of potential biomarkers  as well as for targeted drug delivery . Recent evidences have suggested that EVs play a major role in tissue injury and repair during musculoskeletal diseases. Based on the role of EVs in tissue repair in muscle, it can be speculated that tumour-derived EVs might play a major role in mediating catabolism of distally located tissues. However, it is unclear whether EVs play a major or any role in non-cancer-mediated cachexia (e.g. HIV-associated cachexia). Clearly, additional research is warranted to understand the precise role of EVs in cachexia. Even though significant amount of research is carried out to reduce the burden of cancer-induced cachexia, till date no biomarker has been approved to diagnose cachexia before it makes a significant catabolism of distally located tissues. Knowledge on the role of EVs in the initiation and development of cancer-associated cachexia may aid in the development of new biomarkers and open up novel therapeutic options.
human adipose tissue-derived mesenchymal stem cells
heat shock proteins 70 and 90
mitogen-activated protein kinase
nuclear transcription factor-kappa B
p38-mitogen-activated protein kinase
phosphorylated hormone-sensitive lipase
Toll-like receptor 4
tumour necrosis factor alpha
white adipose tissue
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
S.M. is supported by Ramaciotti Establishment Grant. The authors thank Michael Liem for contribution with a template for Figure 1. The authors also thank the reviewers for the insightful thought and constructive feedback.
The Authors declare that there are no competing interests associated with the manuscript.