Exosomes and ferroptosis: roles in tumour regulation and new cancer therapies

Research on the biological role of exosomes is rapidly developing, and recent evidence suggests that exosomal effects involve ferroptosis. Exosomes derived from different tissues inhibit ferroptosis, which increases tumour cell chemoresistance. Therefore, exosome-mediated regulation of ferroptosis may be leveraged to design anticancer drugs. This review discusses three pathways of exosome-mediated inhibition of ferroptosis: (1) the Fenton reaction; (2) the ferroptosis defence system, including the Xc-GSH-GPX4 axis and the FSP1/CoQ10/NAD(P)H axis; and (3) lipid peroxidation. We also summarize three recent approaches for combining exosomes and ferroptosis in oncology therapy: (1) promoting exosome-inhibited ferroptosis to enhance chemotherapy; (2) encapsulating exosomes with ferroptosis inducers to inhibit cancers; and (3) developing therapies that combine exosomal inhibitors and ferroptosis inducers. This review will contribute toward establishing effective cancer therapies.


INTRODUCTION
Exosomes, which are extracellular vesicles secreted by most cells and are present in many body fluids (Pegtel & Gould, 2019), have complex biological roles in cancers and promote cancer progression (Elewaily & Elsergany, 2021;Kalluri & LeBleu, 2020). For example, exosomes maintain proliferative signalling (Qu et al., 2009), activate invasion and metastasis (Zarin et al., 2021), induce angiogenesis (Li et al., 2021a), and suppress cell death . Exosomes also enhance tumour cell resistance to radiotherapy and chemotherapy, thereby reducing cancer treatment efficacy (Hu et al., 2019). The exosomal regulation of cancer involves multiple mechanisms, which include the ferroptosis regulation (Brown et al., 2019). Ferroptosis is a newly identified iron-dependent regulated cell death (RCD), which is caused by massive lipid peroxidation-mediated membrane damage (Chen et al., 2021d). Inhibition of ferroptosis promotes cancer progression (Xu et al., 2020;Zhang et al., 2021a). Ferroptosis regulation strategies have been applied in radiotherapy (Zhang et al., 2021d) and chemotherapy (Niu et al., 2021) approaches for cancers. This review discusses the effects of exosomes on tumour biological behaviours. We focus on three pathways that mediate exosomal actions on ferroptosis, including the Fenton reaction, the ferroptosis defence system (the Xc-GSH-GPX4 axis, and the FSP1/CoQ 10 /NAD(P)H axis), and lipid peroxidation. We propose strategies for applying these pathways to develop cancer therapies. This review summarizes innovative new strategies for reducing tumour chemoresistance and developing more effective exosomebased cancer treatment strategies that target ferroptosis (Fig. 1).

Why this review is needed and who it is intended for
Exosomes have gained interest recently because of their biological roles in cancer. Recent work has identified a complex relationship between exosomes and ferroptosis. However, no review summarizes in detail the regulatory pathways of cell-derived exosomes on ferroptosis and their potential for cancer therapy. This review discusses the effects of exosomes on tumour cell biology and summarizes three pathways of exosome-ferroptosis regulation. We also propose therapeutic strategies based on the exosome-ferroptosis effect. Our review will appeal to researchers interested in ferroptosis and exosomes, providing them with innovative ideas and insights for future experiments, as well as an overview of research on the combination of exosomes and ferroptosis in tumour therapy.

SURVEY METHODOLOGY
We conducted a systematic search of the literature to identify relevant articles for this review using PubMed, Web of Science, and Google Scholar, with the last search conducted on March 5, 2022. The search was performed in full-text journals, focusing on the regulatory pathways of exosomes on ferroptosis and their role in cancers. The keywords used and their synonyms and variants could be classified into categories and any combination of words from different categories was used for the search. The categories we used are as follows: 1. About exosomes: exosomes; exosomal; extracellular vesicles (EVs); exosomal biosynthesis; secretion; uptake; endocytosis 2. About ferroptosis: ferroptosis; anti-ferroptosis; ferroptosis mechanisms; lipid peroxidation; Fenton reaction; arachidonic acid lipoxygenases (ALOXs); glutathione (GSH); glutathione peroxidase 4 (GPX4); ferroptosis suppressor protein 1 (FSP1); GTP cyclohydrolase 1 (GCH1); BH4; solute carrier family 7 member 11 (SLC7A11); solute carrier family 3 member 2 (SLC3A2); ferroptosis inducers; ferritinophagy; ferroptosis defence; System Xc-3. About tumour: tumour; cancer; anticancer; antitumour; tumourigenesis; invasion; migration; cell proliferation; angiogenesis; metastasis; inflammatory; cell death; apoptosis; chemoresistance; radioresistance; antimicrobial death; immune escape; immunosuppression. The words were merged via the Boolean operators 'AND' and 'OR'. The initial search screened approximately 600 relevant articles written in English that could be useful for this review.

The biological role of exosomes in tumours
Exosomal cargoes include RNA, DNA, proteins, carbohydrates, and lipids. The RNA species include mRNAs, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs) (O'Brien et al., 2020). The biological roles of exosomes have become a topic of interest (Kalluri & LeBleu, 2020), especially the role of exosomes in tumour development and cancer progression (Elewaily & Elsergany, 2021;Pi et al., 2021). The regulation of biological tumour phenotypes by exosomes has mainly focused on lncRNAs and microRNAs, followed by proteins and lipids (Table 1).
of inflammatory mediators, thereby promoting cellular inflammatory responses and tumour progression (Chow et al., 2014;Wu et al., 2016). In contrast, other tumour-derived exosomes attenuate tumour inflammation and promote the immune escape of cancer cells (Othman, Jamal & Abu, 2019). Among these exosomes, programmed cell death 1 (PD-1) and its ligand (PD-L1) are the most well studied (Xie et al., 2019). By binding the PD-1 receptor expressed on activated T cells, PD-L1 inhibits the activation and proliferation of T cells, thereby protecting tumour cells from being killed by T cells and leading to immune escape (Chen et al., 2018;Lawler et al., 2020). Exosomes secreted by tumour cells and cancer-associated fibroblasts (CAFs) promote tumour cell chemoresistance through the delivery of exosomal cargo (Hu et al., 2019;Yang et al., 2020b). A recent study proposes that the CAF-derived exosome miR-522 inhibits ferroptosis by inhibiting the activity of the arachidonate 15-lipoxygenase (ALOX15) and reducing lipid reactive oxygen species (ROS) accumulation and lipid peroxidation, thereby promoting chemoresistance . In addition, exosome-mediated ferroptosis inhibition is a novel mechanism for gastric cancer (GC)-acquired chemoresistance. The mechanism of ferroptosis inhibition will be described in subsequent sections.

Ferroptosis regulation in tumours
Ferroptosis is a form of RCD characterized by ROS accumulation and lipid peroxidation (Dixon et al., 2012). The cessation of lipid peroxide removal triggers ferroptosis (Fig. 2). One of the primary ferroptosis mechanisms includes enzymatic and nonenzymatic lipid peroxidation. Enzymatic lipid peroxidation is an oxidative reaction that occurs in the presence of ALOXs, whereas nonenzymatic lipid peroxidation is driven by iron and ROS-induced free radicals via the Fenton reaction (Chen et al., 2021d;Jiang, Stockwell & Conrad, 2021;Tang et al., 2021). Downregulation of ALOX15 expression and enzymatic lipid production promotes cancer progression (Tian et al., 2017), and activation of ALOX15 in cancer cells inhibits cancer growth (Weigert et al., 2018). ALOX15 catalyses enzymatic lipid peroxidation, suggesting that ALOX15 may inhibit tumours by promoting ferroptosis. Another enzyme associated with lipid peroxidation, stearoyl-CoA desaturase 1 (SCD1), promotes anti-ferroptosis and tumour growth in gastric cancer cells .
Iron metabolism, including Fe 3+ input, Fe reaction, and Fe 2+ output, is another mechanism involved in ferroptosis. Ferritin is the main site of iron storage in the cell, and consists of ferritin light chain (FTL) and ferritin heavy chain 1 (FTH1) (Muhoberac & Vidal, 2019). Iron metabolism may change in tumour cells and promote the initiation and growth of cancer. The lncRNA RP11-89 sponges miR-129-5p and upregulates prominin2 (prom2), thereby promoting iron export and inhibiting ferroptosis to facilitate tumourigenesis (Luo et al., 2021).
Ferroptosis activation inhibits tumour progression. The loss of the Xc − system in melanoma reduces intracellular cystine levels, decreasing GSH synthesis and GPX4 activity. This process promotes ferroptosis and eliminates tumour metastasis (Sato et al., 2020). Gambogenic acid induces ferroptosis via the p53/SLC7A11/GPX4 signalling pathway and inhibits melanoma cell migration and epithelial-to-mesenchymal transition (Wang et al., 2020e). Drug-resistant cancer cells depend on GPX4 and are more likely to undergo ferroptosis (Hangauer et al., 2017;Tsoi et al., 2018). Zinc finger E-Box binding homeobox 1 (ZEB1) has high expression levels in several treatment-resistant cancer cell lines that depend on GPX4 and high sensitivity to ferroptosis caused by GPX4 inhibition (Viswanathan et al., 2017). This suggests that the induction of ferroptosis may be a promising approach for cancer treatment. Induced ferroptosis or ferroptosis inducers combined with chemotherapy or radiotherapy can eliminate and inhibit tumour cells. Erianin induces calcium/calmodulin-dependent ferroptosis and inhibits cancer cell migration, thereby exhibiting anticancer activity (Chen et al., 2020). The P62-Kelch like ECH associated protein 1 (KEAP1)-nuclear factor erythroid-2 related factor 2 (Nrf2) pathway has a role in HCC cell ferroptosis, and inhibition of Nrf2 expression upregulates iron and ROS levels and promotes the antitumour effects of ferroptosis inducers (Sun et al., 2016). Ferroptosis inducers can be combined with radiotherapy in cancer treatment strategies to suppress radioresistant cancers by inactivating SLC7A11 or GPX4 (Lang et al., 2019;Lei et al., 2020;Ye et al., 2020). In a murine xenograft model and human patientderived models, ferroptosis inducers enhance the antitumour effects of radiotherapy (Ye et al., 2020).

Mechanism of exosome-mediated ferroptosis
Exosomes transport specialized cargo molecules that regulate the expression of ferroptosisrelated genes in receptor cells. Since the regulation of ferroptosis affects tumour development, the mechanism of exosome-mediated ferroptosis must also be investigated (Fig. 3).

Exosomes directly inhibit the Fenton reaction
Iron accumulation contributes to ROS production through the Fenton reaction, promoting lipid peroxidation and leading to ferroptosis. Exosomes can reduce the intracellular iron content, which may inhibit the Fenton reaction and subsequent ferroptosis (Dixon et al., 2012).

Molecular mechanisms of exosomes and tumour therapies combining ferroptosis and exosomes
Exosomal regulation of ferroptosis in receptor cells is related to exosome synthesis and uptake mechanisms. There are five key steps in exosomal biosynthesis: (1) endocytosis of the cytoplasmic membrane, (2) early sorting endosomes (ESE), (3) late sorting endosomes (LSE), (4) formation of multivesicular bodies (MVBs) containing future exosomes, and (5) exosome release (Kalluri & LeBleu, 2020;Pan & Johnstone, 1983). These processes involve a variety of proteins and lipids. For example, the Endosomal sorting complex required for transport (ESCRT) proteins bind in a continuous complex (ESCRT-0, -I, -II, and -III) across the MVB membrane to regulate cargo orientation and the formation of intraluminal vesicles (ILVs) (Hurley, 2015). The transmembrane Tetraspanin proteins induce membrane-bending structures and promote exosome formation (Andreu & Yáñez Mó, 2014). Exosome secretion from the cell is mediated by trafficking proteins. Rab GTPase is involved in intracellular vesicle translocation and trafficking MVB to the plasma membrane for exosome release (Hsu et al., 2010;Ostrowski et al., 2010). Inhibition of Rab35 results in the intracellular accumulation of vesicles and reduced exosome secretion (Hsu et al., 2010). The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complex is required for MVB fusion with the plasma membrane (Zhao, Holmgren & Hinas, 2017). Wnt-containing exosomes cannot be secreted without the YKT6 SNARE (Gross et al., 2012). Exosome budding and release may require the actin cytoskeleton and microtubule network (Mathieu et al., 2019).
The combination of exosomes and ferroptosis opens new strategies for cancer therapy. Current research is focused on three different strategies: (1) promoting exosome-inhibited ferroptosis to enhance the effects of chemotherapy; (2) encapsulating exosomes with ferroptosis inducers to inhibit cancers; and (3) developing therapies that combine exosomal inhibitors and ferroptosis inducers (Fig. 4).
In summary, cargoes in exosomes, miRNAs and lncRNAs, are responsible for cancer chemoresistance. Different tumour cell lines have different sensitivities to ferroptosis (Hangauer et al., 2017;Tsoi et al., 2018). A critical mechanism underlying these differences at the cellular level is caused by differences in prom2 expression (Brown et al., 2019). Ferroptotic stress (e.g., interference with GPX4, isolated cells, and extracellular matrix) induces prom2 expression in breast carcinoma cells (Brown et al., 2019). prom2Prom2 promotes the binding of intraluminal vesicles to iron-containing ferritin to form ferritin-containing multivesicular bodies. Transporting ferritin out of the cell via exosomes inhibits ferroptosis (Strzyz, 2020).

Mechanism underlying exosomal inhibition of ferroptosis
The upregulation of GPX4 expression and activity has been reported in ferroptosis, but the effects on other Xc-GSH-GPX4 axis genes, such as SLC7A11 and SLC3A2, have been studied less thoroughly. Exosomal miR-4443 modulates FSP1 m6A modification-mediated ferroptosis and facilitates cisplatin resistance in NSCLC. Furthermore, exosomal miR-4443 may regulate ferroptosis-related genes other than FSP1, so the specific pathways of action remain to be established (Song et al., 2021b). There are no reports on exosomal regulation of the third ferroptosis defence system, the GCH1-BH4 axis. ALOX15, the AMPK pathway, and PRDX6 are involved in the exosome-ferroptosis effect, but the other ferroptosis pathways remain to be explored. Future research should conduct a deep pathway study to achieve a more comprehensive molecular understanding of exosomal inhibition of ferroptosis.

Exosome-based drug delivery systems
Cisplatin-resistant NSCLC-derived exosomal miR-4443 promotes cisplatin resistance in NSCLC by regulating FSP1 m6A modification-mediated ferroptosis (Song et al., 2021b). This suggests that future studies could reduce cisplatin resistance and develop new anticancer strategies by restoring METTL3/FSP1-mediated ferroptosis in tumour cells.
Cells release iron-containing exosomes by expressing prom2, which transports iron out of the cell and thereby suppresses ferroptosis (Strzyz, 2020). Recent studies have attempted to reverse ferroptosis in cancer cells by inhibiting prom2 transcription (Brown et al., 2021). Heat shock factor 1 (HSF1) positively regulates Prom2 transcription. This suggests that Prom2 transcription may be blocked with HSF1 inhibitors, thereby sensitizing chemoresistant cancer cells to drugs that induce ferroptosis. However, the hypothesis still needs to be studied and more prom2 inhibitors must be tested.
Exosomes have become an active topic of current research as a drug delivery system (Patil, Sawant & Kunda, 2020). Studies on exosomes loaded with anticancer drugs targeting ferroptosis are limited to erastin acting on the system Xc − . In the future, additional anticancer drugs may be developed to target different ferroptosis pathways. For example, a first-line therapeutic agent for glioblastoma (temozolomide) may induce ferroptosis by targeting DMT1 expression in glioblastoma cells, which partially inhibits cell growth (Song et al., 2021a).

Exosomes for cancer diagnosis and prognosis
Exosomes have an important role in liquid biopsies for early detection and prognosis prediction of cancer (Li et al., 2021b). Altered ferroptosis markers in exosomes may be useful biomarkers for cancer screening, such as the early detection of HCC (Sanchez et al., 2021) and PC . The lipid composition of HCC and PC cell-derived exosomes is altered, and pathway analysis implicates ferroptosis. Lipidomic profiling in plasma exosomes plays a role in the early detection of HCC in patients with cirrhosis (Sanchez et al., 2021), and molecular lipids in urinary exosomes can be used as biomarkers for PC (Skotland et al., 2017). Ferroptotic pancreatic ductal adenocarcinoma cells (PDACs) with exosomal KRAS G12D may provide information about the prognosis of pancreatic cancer (Dai et al., 2020). Oxidative-stressed PDACs produce autophagy-dependent ferroptosis, releasing KRAS G12D , which is packaged extracellularly as Exo-KRAS G12D (Dai et al., 2020). Exo-KRAS G12D activates signal transducer and activator of transcription 3 (STAT3)dependent fatty acid oxidation pathways, polarizing tumour-associated macrophages to an M2-like native phenotype and leading to poor prognosis in pancreatic cancer patients (Dai et al., 2020). This implies that binding ferroptosis to exosomes holds potential in liquid biopsies of tumours.

CONCLUSIONS
Exosomes play an essential role in tumour regulation, which involves ferroptosis. Different tissue-derived exosomes inhibit ferroptosis via different pathways, which future work must explore. Exosomal inhibition of ferroptosis drives cancer chemoresistance, and new cancer therapeutic agents combining ferroptosis and exosomes have been reported. Future work on exosomes will open new approaches for developing innovative cancer therapies and leveraging exosome-ferroptosis effects. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors: The

Competing Interests
The authors declare there are no competing interests.

Author Contributions
• Yixin Shi conceived and designed the experiments, performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.
• Bingrun Qiu performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.
• Linyang Huang and Yiling Li performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.
• Jie Lin conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.
• Yiting Ze analyzed the data, prepared figures and/or tables, and approved the final draft.
• Chenglong Huang analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.
• Yang Yao conceived and designed the experiments, performed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Data Availability
The following information was supplied regarding data availability: This is a Literature Review.