Exosomes are small biovesicles that originate from the endosomal pathway and are released into bodily fluids when multivesicular bodies fuse with the plasma membrane. They contain cell-specific cargoes, including proteins, lipids, and genetic materials. These cargoes can be absorbed by nearby or distant cells to affect their biological activities. This unique capability positions exosomes as highly promising candidates for use as non-invasive diagnostic biomarkers and therapeutic delivery vehicles [1, 2]. Early studies showed that exosomes originate from inside cells. These studies examined how exosomes help remove proteins from the surface of developing blood cells as they transform into red blood cells [3]. Since exosomes were first discovered in reticulocytes, a type of blood cell, it has been found that many types of cells, especially different kinds of cancer cells, release exosomes into the extracellular space. Exosomes can be commonly found in body fluids such as blood, saliva, and urine [4]. Despite ongoing research, the full significance of exosomes in biology is not yet fully understood. Exosomes are believed to facilitate cell communication by sharing active substances, which can impact the function of target cells and play a role in various bodily functions and diseases [5]. These membrane systems allow large molecules, such as proteins, fats, and DNA, to be transported from one cell to another over long distances without being harmed [6]. The study of exosomes has made significant progress since the 1970s [7, 8]. Pan et al. coined the term "exosome" to describe these particles, which were subsequently characterized as being derived from the endocytic pathway and released by various cell types. In addition, Zitvogel et al. demonstrated that exosomes play a role in antigen presentation to immune cells, suggesting their involvement in intercellular communication [7, 9, 10]. The field gained momentum in the 1990s by discovering exosome involvement in intercellular communication and their potential as therapeutic agents [11]. In the 2000s, isolation techniques and the identification of microRNAs within exosomes. In recent years, exosome engineering and clinical trials have become prominent in research [9, 12, 13]. Current exosome research focuses on developing targeted therapies, integrating exosomes with other technologies, and addressing ethical considerations [14, 15] (Fig. 1).

Fig. 1

A decade of exosome research: from discovery to cutting-edge applications

Source and structure of exosomes inside cells

Exosomes which are approximately 30–100 nm wide [16] are formed through a process called endocytosis. During this process, a paortion of the late endosome membrane folds in, leading to the creation of multivesicular bodies (MVBs) containing small vesicles filled with fluid from the cell (ILVs). The MVB can either merge with the outer cell membrane to release the ILVs as exosomes into the extracellular space or fuse with lysosomes to degrade the contents [2]. The number, generation, and eventual location of ILVs and exosomes are regulated by various cellular processes. These processes collaborate meticulously and are closely managed during the movement of membranes involved in endocytosis [17]. This guid aims to assist researchers in selecting the most appropriate isolation methods for exosomes based on their research objectives and available resources.The creation and release of exosomes are important for making related proteins and sorting their contents [18]. The creation and release of exosomes are also managed by other methods that do not depend on ESCRT [19]. Exosomes are different from other small particles that come from cells because of their source, size, appearance, and composition. Often researchers, identify the type of vesicle by looking at it under an electron microscope and checking for certain specific proteins [20]. All exosomes have specific proteins because they come from endosomes. These include proteins that help move things in and out of cells, like Rab GTPases and Annexins. They also have heat-shock proteins and other proteins that help form multivesicular bodies (MVBs), as well as tetraspanins such as CD9, CD63, CD81, and CD82 [21, 22]. Based on their origin and intended function, exosomes may exhibit unique proteins specific to certain cell types. For example, mature dendritic cell exosomes have a lot of MHC class II and CD86, which aid in the activation of CD4 + T cells. Studies of lipid content have revealed that exosomes contain significant amounts of cholesterol from lipid rafts, along with a type of fatty substance called ceramide and other types of fats [23]. Besides proteins and fats, exosomes can also carry nucleic acids, especially mRNA and microRNA. These can influence how recipient cells express proteins and signal each other [24]. Table 1 shows a summary of different standard methods used to separate exosomes. Exosomes are made by cells that carry different types of molecules. The table shows a summary of different ways used to collect exosomes from biological samples. These techniques include spinning things fast (ultracentrifugation), separating based on how heavy something is (density gradient centrifugation), sorting based on size (size exclusion chromatography), using antibodies to find specific things (immunoaffinity methods), and making things clump together (precipitation methods). Each method is explained, including what is good about it, what it's not so good at, and what kinds of samples it works best with.

Table 1 Different standard methods for separating exosomesRoles of exosomes in intercellular communication

Exosomes can be thought of as small versions of the cells they originate from. They contain a like fluid and have the same orientation as the outer membrane of the cell. This means that the external part of the cell membrane faces outward, displaying specific proteins and receptors that can be used to measure interactions with other cells [25]. Exosomes are small particles that are released from cells and play an important role in the extracellular environment. They are then dispersed to various locations where they interact with recipient cells [26]. The mechanisms by which exosomes connect with recipient cells have been explained through several methods. Exosomes can merge with the outer layer of the target cell, merging their contents with the target cell's membrane. Alternatively, exosomes can activate cell surface receptors by using special lipids and proteins and showing antigens to these receptors [2, 27]. Also, recipient cells might take in exosomes through methods like pinocytosis, phagocytosis, or receptor-mediated endocytosis [28]. It was later discovered that exosomes are integral for displaying antigens and facilitating communication between cells. They serve as crucial carriers in immune responses. [29]. Consequently, novel applications for exosomes are being proposed as researchers study how different cells and tissues produce them. Exosomes might help with blood clotting, moving cells, growing new blood vessels, healing wounds, fighting inflammation, and controlling how cells behave [30,31,32]. Hematopoietic cells, gut cells, fat cells, nerve cells, connective tissue cells, and some cancer cells have all been shown to send out exosomes into fluid outside their cells in lab studies. Many body fluids, like blood, joint fluid, urine, saliva, milk, and fluids from the chest and abdomen, contain a substantial amount of exosomes [33, 34].

The dual role of exosomes in tumor biology

Exosomes, secreted by various cell types, play a pivotal role in tumor biology. These nanovesicles serve as crucial mediators of intercellular communication, facilitating the transfer of molecular signals that can influence cancer progression, metastasis, and immune evasion [27].

Exosomes derived from cancer cells can promote tumor growth and progression through various mechanisms. They can transfer oncogenic factors, such as microRNAs and proteins, that enhance the malignant characteristics of tumor cells. For instance, exosomes can stimulate angiogenesis, facilitating nutrient supply to tumors [35]. Moreover, exosomes can modify the behavior of nearby cells, creating a supportive niche for tumor growth and metastasis [36].

Furthermore, exosomes play a crucial role in metastasis, enabling cancer cells to invade distant organs. They can prepare pre-metastatic niches by altering the extracellular matrix and modulating the immune response. This strategic manipulation of the microenvironment allows for more effective colonization of metastatic sites [37].

Following this, tumor-derived exosomes can suppress anti-tumor immunity by carrying immunosuppressive molecules that inhibit T-cell activation and promote regulatory T-cell expansion. Indeed, this dual role of exosomes in suppressing immune responses while simultaneously promoting tumor growth emphasizes their importance in oncological contexts [38].

Despite their supportive roles in tumor biology, exosomes also offer therapeutic potential. Engineered exosomes can be used for targeted drug delivery or as vehicles for RNA-based therapies. This highlights the need to distinguish between the pro-tumorigenic and anti-tumorigenic roles of exosomes, providing a balanced view of their significance in cancer research [39].

Conclusively, exosomes are powerful modulators of tumor biology, influencing cancer progression, metastasis, and immune evasion. Their dual roles present both challenges and opportunities in the oncological landscape, making them a critical focus for future research and therapeutic strategies [40].

Exosomes from cancer stem cells (CSC-exosomes)

Extracellular vesicles (EVs) can be classified into three types based on their formation and release: exosomes, microvesicles, and apoptotic bodies [41]. EVs produced by CSCs serve multiple functions, including displaying distinct signals on their surface and transporting materials to other cells in the tumor area. It is believed that exosomes released by cancer stem cells (CSCs) contribute to the formation of the pre-metastatic niche. These exosomes increase the ability of CSCs and similar cells to spread cancer to other parts of the body [42, 43].

The cargo of CSC-exosome and their effects

Researchers have identified a type of RNA called H19, which is produced by cancer stem cells (CSCs) and is carried out of the cell in small vesicles known as exosomes. These exosomes are then absorbed by nearby cells, allowing them to take in specific small molecules called miRs, particularly let-7 [44]. Exosomes from breast cancer stem cells have a lot of certain molecules called miRs that are connected to cancer spreading. Another study discovered that exosomes from cancer stem cells made cancer cells more resistant to chemotherapy drugs like doxorubicin and paclitaxel by using miR-155. The resistance to breast cancer therapy is driven by the induction of epithelial-to-mesenchymal transition (EMT) and involves the activation of anti-apoptotic pathways and drug efflux pumps. MiR-155 plays a significant role in EMT and resistance. Exosomes released by cancer stem cells and resistant breast cancer cells carry miR-155, which can be delivered to sensitive cells, resulting in the development of a resistant phenotype. Similar findings were observed in epithelial ovarian cancer cells and gastric cancer cell lines, where exosomes facilitated the transfer of chemoresistance traits through the delivery of miR-155 and the induction of EMT [45, 46]. Exosomes containing miR-30a and miR-222 were found to enhance the aggressiveness of cancer cells in colon cancer stem cells [47]. In the study on CSC-Exosomes from stomach cancer, 11 unique miRNAs were identified. These miRNAs could potentially aid in the diagnosis of metastasis, which occures when cancer spreads to other parts of the body. CSC-Exosomes from gliomas were found to contain high levels of miRNA-21, which in turn increased the production of vascular endothelial growth factor (VEGF) and promoted blood vessel growth [48]. Further analysis showed that Linc01060 was found in cancer stem cells of glioma with low oxygen levels. This molecule triggered processes that promote cancer growth in glioma cells, leading to more serious disease [49]. Additionally, lung cancer stem cell exosomes were found to increase the likelihood of metastasis by upregulating a molecule called miR-210-3p, which affects the FGFRL1 receptor [50]. Researchers found that exosomes derived from stem cells of oral squamous cell carcinoma exhibited elevated levels of miR-21 and reduced levels of miR-34, which contributed to cancer growth. In a separate study, exosomes from gemcitabine-resistant pancreatic cancer stem cells were shown to contain higher amounts of miR-210. This microRNA was transferred to other cancer cells, conferring resistance to the drug. To assess the efficacy of exosomes in drug delivery, several studies have employed techniques such as flow cytometry, confocal microscopy, and quantitative real-time PCR to track the uptake of exosomes by target cells and measure the delivery of encapsulated molecules. For example, used flow cytometry to quantify exosome uptake by tumor cells and analyzed the expression of encapsulated miRNAs using quantitative real-time [51,52,53]. Exosomes derived from cancer stem cells (CSCs) and non-cancer stem cells in prostate cancer contain distinct types of miRNA. Specifically, CSC-derived exosomes are involved in preparing the surrounding environment for future cancer metastasis [54]. For instance, miRNA-19b-3p promotes the growth of new blood vessels, helping the formation of areas where cancer can spread. It also contributes to cancer dissemination and a process known as EMT when present in CSC exosomes in kidney cancer [55]. Additionally, the transfer of lncRNA facilitated the EMT process in papillary thyroid cancers [56]. Table 2 gives a summary of the miRNAs found in exosomes from various types of cancer. These exosomes contain a diverse range of miRNAs that play crucial roles in cancer growth and metastasis. The table presents a comprehensive list of these exosomal miRNAs, arranged by cancer type, offering valuable insights for their potential use in diagnostic tests or as therapeutic targets. This summary underscores the significance of exosomal miRNAs in cancer research and their potential applications in future studies and medical interventions.

Table 2 Exosomal miRNAs in cancers

In this explanation, Fig. 2 shows how cargo is moved by exosomes. The sorting and mixing of cell materials into MVBs are helped by the ESCRT system. The careful addition of parts from secretory cells creates MVBs. These MVBs can join with lysosomes to break down materials or connect with the cell's outer membrane, releasing exosomes into the space outside the cell through a process called exocytosis. Exosomes hold different types of materials, including mRNAs, lncRNAs, miRNAs, proteins, and transcription factors. When exosomes reach their target cells, they can either combine with the cell's outer layer or be taken in by the cell. Using these methods, the proteins and RNA in exosomes can be sent into the liquid inside the cells or become part of the cell membranes. Also, exosomes can talk to target cells using special proteins on their surface, allowing them to connect directly with other cells [29, 93,94,95].

Fig. 2

Moving materials using exosomes. ESCRT sorts and adds content into MVBs. The careful addition of parts from secretory cells leads to the creation of MVBs (multivesicular bodies). MVBs can either combine with lysosomes to break down their contents or merge with the cell's outer layer to release exosomes outside the cell. Exosomes transport RNA molecules (like mRNAs, lncRNAs, and miRNAs), proteins, and other important factors that help in gene activity. Exosomes can either fuse directly with the surface of the target cells or they can be taken in by the cells. Proteins and RNA are sent into the cytosol or the outer layer of other cells through both methods. Exosomes can talk to specific cells directly using special proteins on their surface

Distinguishing characteristics of CSC-exosome

Even though cancer stem cells (CSCs) are difficult to locate within a tumor, scientists are currently investigating the collaborative function of CSC exosomes and tumor exosomes (TEXs). Unlike exosomes made from regular tumor cells, the exosomes from cancer stem cells in human prostate cancer have their unique miRNA, like a high level of has-miR-1307-5p [54]. Exosomes released by normal cells and stem-like cells in stomach cancer exhibit distinct patterns of miRNA expression [48]. The markers present in CSCs contribute more to the spread of cancer compared to those found in regular tumor cells. Both CSC-exosomes and tumor exosomes (TEXs) can help tumors grow [96]. In order to confer resistance to cell death, enhance mobility, and alter the properties of non-cancer stem cells, pancreatic cancer stem cell exosomes deliver the CD44v6 marker to these non-cancer stem cells. It's interesting to see that mice that received injections of colorectal CSC-Exosomes exhibited prolonged presence of neutrophils in their bone marrow, which displayed increased indications of cancer development. More research is needed to clearly explain the differences in markers and biological activities between CSC-Exosomes and non-stem TEXs [97, 98].

The role of CSC-exosomes in cancer environment and growth

The process by which cancer stem cells change into cancer cells and subsequently revert to cancer stem cells is complicated. Exosomes are important for managing the balance between cancer stem cells and regular cancer cells because they help with communication between all the cells in the tumor area. CSCs help cancer grow by releasing signals that keep cancer cells behaving like stem cells. So far, people have mostly assumed that exosomes from cancer stem cells will show this trait and help create an environment that can lead to tumors [52]. Many features of exosomes derived from cancer cells (CDEXs) are similar to the new roles found for exosomes originating from cancer stem cells (CSC-Exosomes). It is important to note that distinguishing between exosomes made by cancer stem cells and similar types found within complex tumor tissues proves to be challenging based on numerous studies. The functions of CSC-Exosomes, which have recently been discovered in a few studies, are explained above about known markers and the cargo they carry [99].

CSC-exosomes and how they affect cancer stem cells

The interaction between cancer stem cells (CSCs) and regular cancer cells, is facilitated by exosomes, which act as carriers of important signals for managing the development of CSCs and the changes in regular tumor cells, helping keep the balance in the cancer environment. In breast cancer, certain cancer cells exhibit stem cell-like behavior and produce exosomes that carry mRNA messages linked to cancer spread and stem cell function. These exosomes promote the growth of tumors in other cells. The Wnt signaling pathway, which is essential for growth, development, metabolism, and the maintenance of healthy stem cells, is also involved in cancer progression. However, when this pathway becomes activated inappropriately, it can lead to tumor formation and impact the renewal and development of cancer stem cells. There is compelling evidence demonstrating fibroblast exosomes induce colorectal cancer cells to exhibit stem cell characteristics, such as the ability to form spheres and grow tumors. They also increase the number of cancer stem cells in colorectal cancers by activating the Wnt signaling pathway. Additionally, exosomes derived from mesenchymal stem cells (MSCs) activate the Wnt signaling pathway, promoting the growth of breast cancer cells. Exosomes from surrounding cells in lymphoma help change side-population cells into non-side-population cells by carrying the Wnt signaling pathway in cells activated by Wnt3a [100, 101].

EMT and CSCs

The formation of pre-metastatic niches and the recruitment of bone marrow cells occur when other cells take in tissue-specific exosomes (TEXs). This process is influenced by the ability of cancer stem cells (CSCs) to self-renew and differentiate into different cell types, which is strongly influenced by a process called EMT [102]. Through EMT, these cells can gain traits similar to stem cells. Transforming growth factor beta (TGF)-β can start a process called EMT. Exosomes from chronic myeloid leukemia carry a substance called TGF-β1 to other cells. This helps the leukemic cells grow and can lead to the formation of tumors. Claudin 7 is sent into less aggressive cancer cells by exosomes made by the cells that start colon cancer. This process helps change these cells [103]. Reports suggest that CSC-Exosomes carry miRs and lncRNAs that control how CSCs release substances and resist treatment [104].

CSC-exosome and the delivery of reprogramming factors

Exosomes help keep cancer stem cells stable by carrying important proteins or by managing how much of these proteins are made in other cells [105]. Abnormal changes in certain proteins that control gene activity in tumor tissues can lead to normal cancer cells becoming cancer stem cells. Exosomes contain miRNAs that play a vital role in regulating tumor cell growth, survival, and tumor formation. For instance, gastric cancer cells release exosomes containing let-7 microRNAs into the tumor microenvironment, promoting cancer aggressiveness and faster growth. Melanoma cells produce a molecule called miR-222 in exosomes, which can make the cancer aggressiveness. Additionally, tumor cell-derived exosomes are rich in miR-21 and miR-34a molecules miR-21 and miR-34a [106].

Effects of CSC-exosome on the immune system in tumor microenvironment

Recent research shows that CDEXs or TEXs help suppress the immune system in tumors [107]. However, we need to exercise caution before assuming that CSC-Exosomes behave similarly in altering the tumor microenvironment [108]. Exosomes released by brain tumor stem cells contain Tenascin-C, which lowers the activity and growth of T cells. Colorectal cancer stem cells release tiny particles that elevate interleukin-1 levels, leading to the support of tumor growth by neutrophils [109]. In another study, exosomes from colorectal cancer stem cells were introduced to dendritic cells (DCs) causing T-cells to specifically target cancer stem cells. Exosomes from glioblastoma stem cells utilize the STAT-3 pathway to convert monocytes from the M1 type to the M2 type, creating an immune-suppressing environment [110]. CSC-Exosomes also impact the presence of programmed cell death ligand 1 (PD-L1) in macrophages and the transformation of monocytes into myeloid-derived suppressor cells. Additionally, cancer stem cells in glioblastoma produce macrophage migration inhibitory factor (MIF), which encourages the development of myeloid-derived suppressor cells (MDSCs), and further weakens the immune system [111]. In a kidney cancer model, CSC-derived extracellular vesicles (EVs) hindered the development of DCs and immune responses from T cells [112]. The interaction between cancer stem cells (CSCs) and exosomes holds promise for developing new cancer treatments that focus on the immune system; however, many questions remain regarding how CSC-Exosomes precisely influence the immune system in tumors [113, 114]. Importantly, the association and impact of CAR T-cells with exosomes must be investigated and also the significance e of hub long non-coding RNAs along with lncRNA-miRNA-mRNA are of great importance [