HSP90 has a molecular weight of approximately 90 kDa, and its structure usually contains α-α or β-β homodimers (33). HSP90 contains three conserved structural domains: the N-terminal domain (NTD), the middle domain (MD), and the C-terminal domain (CTD) (34).

Notably, the N-terminal structural domain binds to ATP and is also known as the nucleotide-binding domain. Unlike prokaryotic cells, there is a short dynamic region of HSP90 in eukaryotic cells that connects the NTD to the MD, which can increase the flexibility and dynamics of this region to cope with the complex environment of eukaryotic cells (35). The MD subsequently regulates the function of HSP90 by binding the γ-phosphate of NTD-specific ATP, thereby activating its ATPase activity (36). The CTD is mainly responsible for protein dimerization (37). In addition, the HSP90 family consists of four main members, including HSP90α, HSP90β, Gp96, and tumor necrosis factor receptor-related protein 1 (Trap1) (34). Different members are distributed in different cellular compartments. Furthermore, HSP90α is an inducible thermoprotein, whereas HSP90β is a constitutive thermoprotein, both of which are located in the cytoplasm. The main difference between HSP90α and HSP90β is the presence or absence of glutamine fragments. Gp96 and TRAP1 are localized in the endoplasmic reticulum and mitochondria, respectively.

HSP70 (HSP72 or HSPA1) is the most important member of the heat shock protein family; it has a molecular weight of approximately 70 kDa and contains more than 20 different proteins with molecular weights of 68, 72, 73, 75, and 78 kDa. HSP70 consists of two highly conserved structural domains: a nucleotide binding site (NBD) at the N-terminal end and a substrate binding site (SBD) at the C-terminal end (42). The HSP70 family consists of at least eight homologous chaperone proteins that are mainly distributed in the nucleus and cytoplasm. The proteins of this family can be divided into three categories: stress-inducible HSP70, constitutive HSP70, and specific proteins localized in different organelles, such as HSP75 (mitochondria) and GRP78 (endoplasmic reticulum) (43, 44). Under physiological conditions, HSP70 is mainly located in the cytoplasm and functions by assisting in the folding of newly synthesized polypeptides, the assembly of multiprotein complexes, and the transport of proteins across the cell membrane. Under severe stress conditions, HSP70 levels increase rapidly in the nucleus, whereas only a small amount of HSP70 is present in the cytoplasm; moreover, when the cell recovery phase begins, HSP70 gradually disappears from the nucleus, whereas the cytoplasm still maintains a low level of HSP70 expression (45). In addition, HSP70 prevents protein aggregation, promotes refolding of misfolded proteins, and solubilizes aggregated proteins. It also acts synergistically to remove aberrant proteins and their aggregates via cellular degradation mechanisms (46).

HSP60, which is also known as HSPD1 or CPN60, is a stressed heat shock protein that is predominantly found in mitochondria (56). Initially, HSP60 was thought to be localized only in mitochondria; however, as research progressed, it was found to be equally present in the cytoplasm, nucleus, and blood circulation. The N-terminus of HSP60 has a mitochondrial localization sequence that contributes to its localization to mitochondria, whereas the C-terminus contains a series of G-repeat sequences (57). Notably, HSP60 usually forms an asymmetric complex with the cochaperone protein HSP10, whereby these proteins work together in the process of folding and in correcting the misfolding of proteins in the cytoplasm (58).

HSP90-Exs have been shown to function as cellular communicators in the tumor microenvironment (TME), which is largely dependent on their specific location on the membrane surfaces of exosomes (63). HSP90-Exs are involved in two mechanisms, including the activation of metalloproteinase-2 (MMP-2) and human epidermal growth factor receptor-2 (HER-2) and the activation of fibrinogen during tumor metastasis. The precise molecular mechanism by which HSP90-Exs activate MMP-2 is not clear; however, HSP70 is clearly an indispensable coinitiator (64). Notably, HSP90 directly interacts with HER-2 and activates HER-2 downstream molecular pathways to direct cytoskeletal rearrangement and enhance tumor cell invasive properties (65). In addition, it has been reported that breast cancer and glioblastoma cells secrete HSP90-Exs to promote the conversion of fibrinogen to the active form of fibrinolytic enzymes. Specifically, HSP90, protease tissue plasminogen activator (tPA) and membrane-associated protein II are a set of ternary complexes on the surfaces of HSP90-Ex membranes that aid in plasminogen activation (66, 67). The active protease form of fibrinolytic enzymes reprograms the local matrix of the TME and increases the probability of tumor cells in binding to migratory attachment sites. Interestingly, high levels of HSP90-Exs were detected in metastatic oral squamous cell carcinoma (OSCC.) However, the knockdown of HSP90 alone did not significantly reduce tumor metastasis. Both HSP90α and HSP90β must be knocked down via small interfering RNA (siRNA) to effectively inhibit tumor metastasis (68).

Recent studies have shown that HSP90-Exs, which are abundant in the TME, can induce M2-type polarization of macrophages by mediating CD91 on the macrophage surface. This process promotes the progression of pancreatic ductal adenocarcinoma (PDAC), as M2-type macrophages play an important role in the construction of the immunosuppressive tumor microenvironment (69). Similarly, the depletion of HSP90α/β and cell division control-37 (CDC37) ternary complexes via siRNA in OSCC significantly reversed the outcome of tumor progression. This is due to the ability of the ternary complex to encourage the chemotactic M2-type polarization of macrophages, as well as the epithelial-mesenchymal transition (EMT) (70). In addition to the negative immune response triggered by the chemotaxis of M2-type macrophages, HSP90-Exs have also been shown to play a role in activating dendritic cells (DCs) to initiate an adaptive immune response. Studies have shown that HSP90-Exs are able to activate DCs via the nuclear factor kB (NF-κB) pathway, which promotes antigen-presenting ability and costimulatory molecule expression, thereby exerting antitumor immunity (71). In addition, the release of HSP90-Exs by human hepatocellular carcinoma cells has been shown to activate NK cell cytotoxicity, as well as granzyme B production, which is an important factor in the development of a positive tumor immune microenvironment (72). The specific effects of HSP90-Exs on tumor progression are summarized in Figure 1 .

HSP70 cannot leave the cell via the traditional transport pathways of the endoplasmic reticulum and Golgi proteins because it does not have a homologous transmembrane sequence (73). However, HSP70 is able to separate from the cell via other pathways that are related to the environment in which the cell is located and the signaling pathways that are activated. One mechanism involves tumor cells secreting HSP70-carrying membranous vesicles via plasma membrane vesicles, which are usually 40–150 nm in diameter; these vesicles are also known as tumor-derived exosomes (TDEs), and they play a communication role in regulating the TIME (74). Notably, HSP72 on the surfaces of tumor-derived exosomes (TDEs) can produce IL-6 to trigger Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner, which subsequently inhibits T-cell differentiation. However, exosomes from the 3T3 cell line (normal cell line) have no immunosuppressive function. Therefore, this mechanism is sufficient for HSP-Exs to have the specific ability of editing the TIME (75).

Membrane-bound HSP70 (mHSP70) released from tumor cells under the influence of interleukin-2 (IL-2) has been shown to activate natural killer (NK) cells (76). When NK cells are activated, they produce granzyme B, and mHSP70 facilitates the uptake of granzyme B by HSP70-positive tumor cells in a nonperforin-dependent manner. This process subsequently triggers apoptosis via a cysteine-dependent pathway. Intriguingly, researchers cocultured human NK cells with peptides that are present in the external environment of tumor cells (e.g., full-length heat shock protein 70 or a 14-amino acid peptide derived from the C-terminal structural domain of HSP70), which activated NK cells (CD57+/CD94+ NKs) and induced the migration of these cells to recognize and destroy the membranes of HSP70-positive tumor cells (77).

It is promising that exogenous heat shock protein 70 (eHSP70) can send early warning signals to the innate and adaptive immune systems and play a key role in the remodeling process of the TIME. eHSP70 interacts with CD14 and Toll-like receptors 2 and 4 (TLR2/4) to activate NF-κB and prompts antigen-presenting cells (APCs) to release proinflammatory cytokines such as nitric oxide (NO), IL-1β, IL-6, IFN-γ, and TNF-α (78–80). From another perspective, it is possible that HSP70/Bag-4 surface-positive TDEs released by human pancreatic and colorectal cancer cells trigger innate immune responses by activating NK cells (81). NK cells treated with full-length HSP70 and exposed to TKD peptides exhibited effects similar to those described in previous studies (82).

Notably, eHSP70 can trigger IFN-γ production by NK cells, and this induced effect is based on an interactive exchange between NK cells and DCs. This interaction occurs via the binding of the NK cell activation receptor NKG2D to the NKG2D ligand known as MHC type I chain-associated protein A (MICA) on the surface of DCs. Furthermore, eHSP70 has the ability to prompt DCs to express MICA (83). In addition, HSP70 can also induce DC maturation by increasing the expression of MHC class II molecules and other costimulatory molecules (such as CD86, CD83, and CD40). In this process, peptides 407–426 in the C-terminal structural domain of HSP70 are involved in cytokine production (84).

Following activation, mature CDs interact with CD8+ cytotoxic T lymphocytes (CTLs) to trigger and generate an adaptive immune response.

Tumor antigen-peptide complexes that bind to eHSP70 interact with receptors on the surfaces of APCs, including dendritic cells, macrophages, and monocytes, as well as endothelial and epithelial cells. These receptors include CD36, CD91, CD14, CD40, and TLR2/4, as well as scavenger receptors such as LOX-1, SR-A, SREC-1, and FEEL-1. Recently, these receptors have been identified as being possible binding sites for eHSP70 (85). HSP70 receptor-mediated endocytosis is facilitated by this interaction, which results in the interaction of the antigen with the major MHC class I molecules in the histocompatibility complex for cross-delivery, thereby triggering an antitumor immune response (86, 87).

Notably, the substrate-binding domain (SBD) of HSP70 plays a key role in the transport of antigens and their binding to MHC class I molecules (88). Other studies have shown that eHSP70, which does not bind to peptides, triggers a cytotoxic response in helper T-cells, thereby stimulating an adaptive immune response (89). eHSP70 not only transmits stimulatory immune signals as autocrine and paracrine signals in TDEs but also plays a role in tumors, the immune system, endothelial cells, and epithelial cells. These signals can stimulate the inhibitory mechanisms of the immune system, create an inflammatory microenvironment, trigger the activation of MMPs, and accelerate blood vessel formation, thereby accelerating the growth and spread of tumor cells.

eHSP70 has been shown to have significant immunosuppressive effects on myeloid-derived suppressor cells (MDSCs) in experimental models in both mice and humans. From a macroscopic perspective, free eHSP70 has been shown to increase the immunosuppressive capacity of CD4+CD25+FoxP3+ regulatory T-cells (Tregs). This process results in a decrease in the levels of proinflammatory cytokines such as IFN-γ and TNF-α and an increase in the secretion of suppressive cytokines such as IL-10 and TGF-β (90). In addition, eHSP70 can also be regarded as a threat-associated molecular pattern (DAMP) that triggers increased cytokine production, which correspondingly creates an inflammatory microenvironment that contributes to tumor development (91). For example, HSP70 released from heat shock-treated A431 squamous cell carcinoma cells is able to activate the epidermal growth factor receptor (EGFR) and ERK1/2 signaling pathways via interaction with TLR2/4 (92). When considering the key role that epidermal growth factor receptors play in MMP activation and subsequent tumor invasion and metastasis, eHSP70 is likely involved in these biological processes (93). The NF-κB pathway triggered by eHSP70 binding to TLR2/4 on H22 hepatocellular carcinoma cells also has the potential to exhibit antiapoptotic properties that promote tumor growth (94). More specifically, eHSP70 accelerates tumor growth and progression by inducing the release of highly migratory group protein B1 (HMGB1) and the upregulation of MMP-9 in H22 hepatocellular carcinoma cells (95). Similarly, eHSP70 induces MMP-9 expression through the activation of NF-κB and activator protein-1 (AP-1) in human U937 monocytes (96). The specific effects of HSP90-Exs on tumor progression are summarized in Figure 2 .

Recent scientific studies have indicated that the exosome HSP60 has significant antitumor effects in a mouse B-cell lymphoma/leukemia tumor model (A20). In addition, exosomes isolated from heat shock lymphoma cells contain significantly more HSP60 than those produced by unstimulated cells (97). Increased HSP60-Ex levels result in an increased number of molecules associated with immunogenicity, including MHC class I, MHC class II, CD40, CD86, RANTES, and IL-1β. These HSP-Exs are able to more efficiently induce the phenotype and function of DCs, as well as CD8+ and CD4+ T-cells, to reach a mature state, which is essential for triggering tumor rejection. When investigated in a model of hepatocellular carcinoma and colorectal cancer, HSP60-Exs have the ability to stimulate cytotoxicity and granzyme B production in NK cells (72, 98).

HSP60-Exs are also affected by chemotherapeutic agents such as SAHA (serotonin linoleate), which is a histone deacetylase inhibitor (HDACi). SAHA may cause an increase in the secretion of HSP60-Exs, which is paralleled by a decrease in the cytoplasmic concentration of HSP60 (99). Nitrosation of HSP60 is caused by oxidative stress triggered by SAHA, and the molecular chaperones that are involved in nitrification are recognized in exosomes (100). Although nitrated HSP60-Exs can promote the immunogenicity-enhancing effect of SAHA on cancer cells, the molecular mechanisms that are involved in this effect remain to be elucidated (101). As observed with SAHA, the hepatocellular carcinoma cell line HepG2 demonstrated increased HSP60-Ex secretion after treatment with irinotecan HCl and carboplatin. These exosomes induce a stronger antitumor immune response than do those released from untreated cells (72).

Heat shock proteins possess four modes of application in tumor vaccines: 1) exogenous heat shock proteins act as classical exogenous antigens due to the inconsistency of their sequences with those of the host cells; 2) they are reactive because tolerance to self-heat shock proteins is broken or not yet fully established; 3) cross-reactivity between self-heat shock proteins and related proteins triggers an immune response to the latter protein type in the organism; and 4) HSP-antigen complexes induce an effective immune response (i.e., heat shock proteins act as carriers to facilitate the transport of antigenic substances). Currently, the fourth mode of cancer vaccine development is the most promising mode. The fourth mode is specific and capable of generating protective immunity against autologous (but not homologous) tumors. HSP-peptide complexes isolated from healthy individuals fail to effectively trigger tumor immune responses (115). From the perspective of tumor types, cancers have unique antigenic properties. Individual cancers can be vaccinated against their own tumors with specific vaccines; however, such vaccines are not effective against other types of cancers. Therefore, heat shock protein-peptide vaccines that are designed for specific cancers are only effective against the same type of cancer (116). After the vaccine enters into an organism, the organism begins to present the antigen. The binding of APC to the heat shock protein-peptide complex involves multiple receptors and is saturable (117). For example, CD91 (an LDL-associated protein) (118), LOX-1 (a low-density lipoprotein) (119) and Toll-like receptors and their cofactors CD14 and CD40 have been shown to be involved in the binding of heat shock protein-peptide complexes to APCs (120). HSP binding to these receptors promotes APC maturation (121). Although HSP is considered an “exogenous antigen”, it can be converted to the MHC class I pathway via the endogenous MHC class II pathway, which is a process known as “antigen cross-presentation” (86). A clinical study revealed that CD8+ T-cells are required for effective tumor rejection (122). Exogenous antigens are usually presented via MHC class II molecules; however, heat shock proteins are able to enter into the endogenous pathway and consequently be presented to MHC class I molecules (123). Numerous studies have shown that macrophages take up antigens via specific mechanisms. When a macrophage binds to an HSP complex, the HSP acts as a carrier to separate from the peptide complex and is processed and presented in the context of the macrophage’s own MHC class I molecules. Even if only 1% of the specific antigenic peptides are efficiently processed, approximately 107 specific antigenic peptides can still be obtained. These antigenic peptides are sufficient to stimulate T-cells, which results in strong stimulation of the immune system, thus resulting in the unusually high specificity and immunogenicity of the HSP peptide complexes (115). The binding of macrophages to a specific CD8+ T-cell epitope stimulates T-cell clones that are specific for that epitope (86). After vaccination with the HSP peptide vaccine, vaccination with the HSP peptide complex was shown to induce long-lasting memory T-cell immunity, as well as resistance to radiation, which represent characteristics of an idealized vaccine with long-term immune memory function (124).

HSP-Exs are membrane vesicles containing HSPs released by tumor cells. These exosomes are capable of carrying a variety of biologically active molecules, such as antigens, lipids, miRNAs and immunostimulatory factors. As ideal carriers in immunotherapy, exosomes have good biocompatibility. Exosomes function in a relatively gentle manner with cells, thus reducing adverse reactions to the body compared with traditional drug transport carriers (130). HSP-Exs are involved in the regulation of immune activity via multiple mechanisms, including 1) antigen presentation, 2) cytokine release, and 3) regulation of the TME. Based on the abovementioned properties, HSP-Exs can be used as ideal tumor vaccines. The production of the HSP-Ex vaccine is mainly elicited via heat shock treatment of tumor cells to collect and obtain exosomes in the culture medium, after which filtration is performed (such as fractional ultracentrifugation, polymer precipitation and membrane filtration) to efficiently purify the exosomes. The transfection of specific antigen genes into tumor cells via genetic engineering to obtain exosomes that are enriched with the target antigen is another methodological approach (131).

Compared with other biomimetic cancer nanovaccines, the HSP-Ex vaccine contains a broad spectrum of antigens, thus making it more difficult for tumor cells to evade immune surveillance (137). Exosomes have many advantages as antitumor vaccines: 1) the composition of exosomes can be determined, and MHC class I and class II contents can be measured; 2) they can be cryopreserved for at least 6 months; and 3) they can efficiently present antigens to APCs to initiate and amplify antigen-specific immune responses (15). Many studies have demonstrated that HSP-Exs have a promising future as tumor vaccines, and several HSP-Ex immune vaccines have been developed for different tumors.

CD4+ T-cells play a major role in preventing tumorigenesis, whereas CD8+ T-cells are the main antitumor cells. HSP-Exs can induce a significant increase in antitumor immune responses in preventative and therapeutic lymphoma experiments in vivo (138). For example, Florencia et al. targeted exosomes isolated from the ascites of very aggressive murine T-cell lymphomas. These exosomes were experimentally observed to be enriched in HSP70 and HSP90, and these exosome vesicles were then inoculated into naive homozygous mice. The results of this study revealed that these exosomes induced humoral and cellular immune responses (especially specific immune responses) with concomitant induction of immune memory (139). In addition, HSP70-enriched exosomes have been shown to activate the immune system and suppress tumor cells to a greater degree than other exosomes in fibrosarcoma (140). These findings demonstrate that HSP-Ex inoculation can stimulate specific immune responses in the body. Therefore, the use of HSP-Exs as a tumor vaccine can activate the body’s immune system and reprogram the TIME to inhibit tumor progression.

Thermotherapy is now becoming known as a new therapeutic trend due to the limitations of conventional treatments in patients with breast cancer. Heat-stressed tumor cells release exosomes (HS-TEXs) containing carcinoembryonic antigen, HSP70 and MCH I to activate DCs (141). Based on these findings, Sen, Kacoli et al. investigated the secretion of HSP70-Exs in breast cells after thermotherapy and reported that exosomes secreted by breast cancer cells increased the HSP70 content under the stress-inducing conditions of thermotherapy (142). Intriguingly, HSP70 or gp96 stimulates macrophages to induce nitric oxide synthase (iNOS) expression, thus resulting in M1 macrophage polarization (79). Based on the abovementioned results, thermotherapy combined with HSP70-Ex vaccine therapy may have a more comprehensive therapeutic effect on breast tumors. In addition to endogenous HSP-Exs, there is a growing trend toward mimicking the HSP structure in vitro to activate the body’s immunity. In this manner, antigens can be efficiently captured and transported to DCs, thus activating antitumor immunity (143). All of the abovementioned studies demonstrated that tumor-derived HSP-Exs have a good ability to promote the tissue immune response. In addition, the feasibility of the use of HSP-Exs as tumor vaccines has been demonstrated in the context of biomaterial synthesis.

Currently, in vitro modifications of exosomes for cancer treatment are also receiving widespread attention. Modifications of exosomes at the nanometer level can achieve targeting effects, which provides a basis for the synthesis of HSP-Ex vaccines. Using glioblastoma-derived exosomes as a drug delivery system, researchers have synthesized biologically safe exosomes known as “Exo&TDPs”, which can cross the blood-brain barrier and deliver the chemotherapeutic drugs temozolomide (TMZ) and adriamycin (DOX) to glioblastoma tissues. The activation of antitumor immunity kills tumor cells (144). The abovementioned studies demonstrated the high degree of plasticity of tumor-derived exosomes and the possibility of encapsulating HSPs in exosomes for the production of HSP-Ex-based tumor vaccines (145, 146). The development of biologically self-assembled tumor cell-derived cancer nanovaccines offers a novel avenue for exploring ideal cancer immunotherapy. In another study, researchers introduced the ER stress inducer α-mangostin (αM) into melanoma cells via poly(D,L-propylidene-coglycolide) nanoparticles. A biologically self-assembled tumor cell-derived cancer nanovaccine (αM-EX) was subsequently harvested based on the biological process of extracellular throughput of the nanoparticles by the tumor cells. Via ELISA, researchers reported that the expression of tumor antigens and HSP70s was increased in αM-EX and that HSP70 significantly reduced the expression of tumor metastasis-associated proteins. HSP70- rich αM-EX have a strong ability to inhibit tumor growth, which is primarily achieved in the following ways. αM-EX possess a considerable quantity of 70 kDa heat shock proteins (Hsp70s), which are induced by ER stress. These proteins serve not only as endogenous adjuvants but also enhance LN targeting and DC internalization. After injection, αM-EX effectively migrates to LNs and is rapidly endocytosed by DCs, synchronously delivering tumor antigens and adjuvants to DCs, and then strongly inducing an anti-tumor immune response and establishing long-term immune memory. Additionally, the natural targeting of APCs by HSP70s is able to activate the endocytosis of bone marrow-derived dendritic cells (BMDCs) via the presentation of potential antigens and adjuvants (147). Exosome nanostructures enriched with HSPs can enhance the immune system’s response to tumors, whereas the adjuvant HSP70s contained in this material are an endogenous component of the cells, thus eliminating the risk of toxicity from exogenous adjuvants. Similarly, in the synthesis of vaccines for the treatment of established HPV lesions and malignant tumors, researchers have utilized various HSP27 proteins, including recombinant HSP27-E7 protein, tumor cell lysate (TCL-HSP27-E7), and HSP27-E7-Ex. The results showed that all three compounds induced an immune response in the body. Crucially, the presence of higher levels of granzyme B (which is secreted by cytotoxic lymphocytes) in the HSP27-E7-Ex regimen was able to target mitochondrial respiratory chain complex I, thus leading to ROS-dependent cell death (148). Thus, the HSP27-E7-Ex regimen has better tumor killing ability, in addition to a greater safety profile, and is considered to be the most promising strategy for HPV vaccination (149).

The abovementioned studies illustrate that the development and application of HSP-Exs as tumor vaccines are promising. Notably, despite the ability of HSP-Exs to activate antitumor immune responses, their use for tumor therapy is still not completely reliable. This is due to the fact that, in addition to this function, HSP-Exs have a series of contradictory functions, such as recruiting immunosuppressive responses, providing an inflammatory microenvironment, and activating MMPs and angiogenesis to promote tumor cell progression and metastasis. The ability of HSP-Exs to promote apoptosis and tumorigenesis has been demonstrated in different experimental models. Therefore, in the process of using HSP-Exs as tools for actual tumor therapy, a strategy should be selected according to the specific type of cancer, and the elucidation of the immunostimulatory mechanism of exosomes, which ensures that HSP-Exs can be carriers of antigens and adjuvants for cancer vaccines, is crucial.

Clinical studies have detected high levels of HSP90-Exs in bodily fluid samples from patients with melanoma (150), papillary thyroid tumors (151), and rectal cancer (152). A cohort analysis was performed on more than 1,500 participants recruited from hospitals, which included patients with different types of cancer (including liver, lung, breast, colorectal, gastric, pancreatic, and esophageal cancers and lymphoma), as well as healthy individuals. The results showed that the detection of HSP90-Ex levels in plasma could identify 80% of cancer patients, even in the early stages of cancer development. HSP90-Ex is particularly important for cancer diagnosis in patients with high expression of ADAM10, which is a surface protein encoded by the chromosome 15 gene (153). Clinical studies have demonstrated that HSP-Exs can be used as circulating markers to predict the probability of cancer in patients.

In contrast, HSP70-Exs are also clinically used for disease diagnosis; for example, HSP70-Exs can be used for the early diagnosis of cancer. Related studies have revealed that noncancer cells (normal cells) release exosomes that do not express HSP70; however, tumor cells release high levels of exosomes and express HSP70 on their membrane surfaces (154). This study analyzed HSP70-Ex in combination with currently used biomarkers, including carcinoembryonic antigen (CEA) and carcinoembryonic antigen 19-9 (CA19-9), by measuring HSP70-Ex levels in a group of patients with lung cancer and in a group of healthy patients; the results suggested that HSP70-Ex can be used for the initial diagnosis of early-stage lung cancer (155). HSP70-Ex is a superior biomarker for predicting tumor metastasis compared with circulating tumor cells (CTCs), which represent a current clinical diagnostic and detection indicator for predicting cancer metastasis (156). This benefit is due to the availability of exosomes with a uniform systemic distribution, a wide detection range and strong protection (157). The presence of HSP70-Exos may additionally serve as a predictor of the response to treatment. During the immunophenotyping of peripheral blood lymphocytes via multiparametric flow cytometry, HSP-Ex levels were found to be sequentially elevated with increasing tumor stage and metastasis. Notably, HSP70-Ex was shown to be persistently low when the patient’s condition was stabilized. In addition, studies have demonstrated that HSP70-Exs are associated with prognostic survival in a variety of cancers, including lung cancer, head and neck squamous cell carcinoma, gastric cancer, breast cancer, and glioblastoma (158–162). HSP60-Ex homeostasis is a key factor for the pathogenesis of cancer. Increased protein levels of HSP60 have been detected in specimens of solid tumors such as breast and colon cancers; therefore, HSP60-Ex has great potential as being an indicator of cancer diagnosis and prognosis (163).