### Exosomal Heat Shock Proteins as Biomarkers of Cancer Progression and Efficacy of Anticancer Therapy  
Exosomes are nanometer-sized bioactive vesicles secreted by a diverse range of cells and present in body fluids, such as: blood, urine, cerebrospinal fluid, breast milk and saliva, as well as bronchoalveolar lavage, ascitic and amniotic fluids [84]. Exosomes under an electron microscope have a \"cupped\" or \"saucer-like\" morphology [85, 86]. There are various validated methods for exosome extraction. In most studies, exosomes were isolated from various cell lines [87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101], autologous tumor cells [99], blood [95, 100, 101], urine [95], or ascites samples [92, 102]. Supernatants were separated by serial centrifugation at low speed to eliminate cellular debris and filtration [87, 88, 89, 90, 95, 99, 100, 101]. Then, exosomes were ultracentrifuged and washed in phosphate-buffered saline solution. The assessment of the exosomal fraction was performed by electron microscopy [87, 89, 93, 97, 99, 100, 102], Western blot analysis [87, 88, 89, 96, 98, 99, 100, 103], Bradford assay [89, 91], bicinchininic acid (BCA) assay [92, 94], NanoSight LM10 tracking system [95, 97], or acetylcholinesteraseMessage assay [100]. The molecular content of exosomes reflects the specialized functions of the original cells. However, the fate of exosomes remains enigmatic. They are able to bind target cells and/or exchange molecules, thus modulating the activity of other cells. Many families of proteins are expressed on the surface of exosomes, such as targeting/adhesion molecules, membrane and cytoskeleton molecules, Hsps and signal transduction proteins [104, 105, 106]. It has been demonstrated that various cancerous cells release exosomes containing Hsps--either by passive release, e.g., from damaged, stressed, or dead cells, or active release, including secretion of Hsp-containing exosomes [91, 107, 108, 109]. It has been hypothesized that pathological conditions, such as fever and acidosis, would increase the leakiness of exosomes and release of Hsps e.g., Hsp60 [110]. As reported by the [PERSON] group, different stimuli will result in different proteins being released in exosomes from the same cell type. They showed that exosomes may form in the multivesicular body, which either traffics to the lysosome or fuses with the plasma membrane, emptying its contents of exosomes into the extracellular space [110]. However, the regulation of the release of exosomal Hsps, remains to be elucidated. These exosomal Hsps play many important roles in cancer, including: immune system regulation [91, 111, 112], cell differentiation [113, 114], vascular homeostasis [109], angiogenesis, epithelial-mesenchymal transition (EMT), cell migration, invasion and metastasis formation [76, 88, 115, 116, 117, 118, 119]. Moreover, exosomes also contain lipids, and various types of RNA and DNA, which may be used for malignant growth and dissemination [120, 121, 122, 123, 124, 125, 126]. Several studies have reported that elevated levels of Hsps can protect malignant cells against therapy-induced apoptosis [3, 127]. It has been suggested that exosomes released by tumor cells are essential factors involved in the resistance-associated secretory phenotype (RASP) of cells. Molecular co-transfer of Hsps with oncogenic factors to recipient cells can promote cancer progression and resistance against stresses such as hypoxia, radiation, drugs, and immune systems. In addition, the RASP of tumor cells can eject anticancer drugs, targeted therapeutics, and immune checkpoint inhibitors with oncogenes [128]. Recent studies have also confirmed that Hsps localized on the surface of exosomes, secreted by normal and tumor cells, are the key players in the intercellular cross-talk during the course of cancer. It has been reported that microvesicles expressing Hsp70 on their surface activate immune system cells, including natural killer (NK) cells [91, 129] and macrophages [130].