**7. Role of extracellular vesicles in cancer prognosis and treatment response**

An increasing amount of research has established that EVs are present in every human biological fluid including lymphatic and seminal fluids, bile, urine, breast milk, ascites, cerebrospinal fluid, saliva and blood, making these fluids a good source for many liquid biopsy approaches [120–122]. An increase in the rate of release of EVs on the account of cellular activation and/or during pathologic conditions may be considered an indication of a possible pathologic condition [123–125]. Real-time cancer treatment response and monitoring can be done using cancer-derived components obtained from these body fluids. The components include EVs, microRNA, circulating tumor cells (CTC), circulating cell-free tumor DNA, long non-coding RNA and EVs [126]. During the development and treatment of cancer conditions, the state of the cell is revealed by the level of active secretion of EVs, which provide timely information on the changing dynamics of the cell [18, 127]. Cancer-derived components like miRNAs obtained through liquid biopsy inhibits mRNA degradation by binding to coding sequences, 5'untranslated region (UTR), or 3'UTR of target mRNAs leading to the inhibition of mRNA degradation or translation [128]. When miRNAs bind to target mRNAs, the mRNA level as well as protein expression are essentially regulated. This means, circulating EVs are latent tools that are utilized in the quest to find a way of monitoring changes in tumor cells during treatment.

A number of studies have reported the relationship between EVs and cancer treatment response. The presence of immune checkpoints and the application of the blocking of these points by some drugs have been exploited in novel anti-cancer treatment regimens [129]. Research into the capacity of EVs as a regulating tool for checkpoint therapy has contributed immensely to the growing need of the essence of monitoring immunotherapy. Anti-tumor immunity and related expressions can be suppressed by programmed cell death 1 ligand (PDL-1) and the identification of these ligands on EVs has shown the potential for use as biomarkers in tumor patients [130]. In a syngeneic mouse melanoma model in C57BL/6 mice and B16-F10 cells experiment by Chen et al. [131]. Analysis of PDL-1 expression proved the application of EVs as a potential monitoring tool in PDL-1 therapy in melanoma patients. PDL-1 expression was either present or knocked down in these models and the levels of tumor-infiltrating CD8+ T-lymphocytes was significantly reduced in the PDL-1

expressing group compared those knocked down. A positive correlation which varied all through anti-PDL-1 therapy was observed of interferon-γ and the level of EV associated PDL-1 during the analysis of patients with metastatic melanoma [131].

In some specific cancer studies, König et al., [132] analyzed EV concentration and circulating tumor cells in breast cancer patients as a marker for the close observation, monitoring and prediction of prognosis in primary and locally advanced breast cancer. Analysis of the cells and EVs were done before and after the administration of neoadjuvant chemotherapy (NACT) prior to a surgical procedure. Patients' response to NAC is an early indication of the efficacy of subsequent systemic therapy. The overall after-NACT response is a strong prognostic factor for the risk of reoccurrence [133]. Patients with a pathologic complete response (pCR) after NACT have a significant higher overall as well as disease-free survival (OS, DFS) than their counterpart patients with residual invasive disease [134]. Studies have shown that before the administration of NACT during therapy, there is an overall an increase in EV concentration, which is linked to lymph node infiltration, while the after-NACT elevation of EV concentration is associated with reduced three-year progression-free and overall survival. This means, the analysis of EVs together with CTC analysis is a promising tool in the assessment of residual disease and the monitoring of therapy and disease outcome [132]. Other studies have used EVs in diverse ways with respect to their role in treatment response and prognosis. The first exosome-based liquid biopsy test, ExoDx™ Prostate IntelliScore (Exosome Diagnostics, Inc., Waltham, MA, USA), was approved by the Food and Administration Authority (FDA) in 2019 to analyze the exosomal RNA for the biomarkers PCA3, TMPRSS2:ERG, and SPDEF on urine specimen [135]. The prostate specific antigen available in this approach is an effective diagnostic and prognostic tool and the monitoring of this antigen together with digital rectal examination is utilized in men who have gone through a definitive therapy for localized cancer of the prostate. Again, in non-metastatic prostate cancer patients undergoing radiotherapy, a higher concentration of circulating EVs have been detected by Nano tracking analysis as a means of monitoring treatment response [136]. The study proposed a possible radiation specific induction resulting from the upregulation of hsa-miR-21-5p and hsa-let-7a-5p, both of which are specific miRNAs related to prostate cancer and radiotherapy [137]. This is further supported by the observation of altered expression of blood extracted EVs and their miRNA cargoes in the monitoring of prostate cancer radiotherapy response [138]. A high expression of some specific miRNAs before radiotherapy were noted to be an indication of better therapeutic outcomes [138]. More applications of the role of EVs in the cancer are recorded for cancer conditions such as glioblastoma [139], colorectal [140], liver [141], and non-solid cancers [142].
