**4. Cancer immunotherapy biomarkers and challenges**

Tumor-derived extracellular vesicles, which transfer immunosuppressive chemicals like PD-L1, TGF1, FasL, TRAIL, and NKG2D ligands, are the major transporters of tumor immune evasion and potential therapeutic targets. Anti-programmed death-1 (PD-1) and its ligand (PD-L1) antibodies are immune checkpoint inhibitors (ICI), stimulating an antitumor immune response over blocking inhibitory immune signaling. Immune checkpoint inhibitors therapy has become potential in determining effectiveness in different cancers such as non-small cell lung cancer, head, and neck squamous cell carcinoma, melanoma, etc. [39]. Un happily only a few patients have responded to the checkpoint inhibitors and there is a critical need to find out the reason for an adaptive immune response [40].

PD-1 and PD-L1 are located on the exterior of the tumor cells, and it acted as a second signal when it is attached to the PD-1 receptors on T-cells. There are a few blocking antibodies that target PD-1 and PD-L1, like pembrolizumab (Merck) and nivolumab (Bristol Meyer-Squibb) enhance the anti-tumor immune response by opposing this inhibitory signal [41]. The response rate percent is depending on the type of tumor and is a distinctive group of patients gained a very less response. PD-L1 as a biomarker and its expression levels on tumor cells were studied using immunohistochemistry has shown to act as a prognostic marker as well prognostic to anti-PD-1 therapy [42]. The tissue-based testing method needs a decent tissue biopsy. A tissue biopsy test method also has a few advantages like bleeding, infection, and other procedural complication like causing pneumothorax in the case of parenchymal lung biopsy. Tissue can be isolated either from the primary tumor or a metastatic tumor lesion upon the different factors that are considered. Hence the expression levels may differ based on the tumor tissues isolated. Researchers have also seen [43]. These results of PD-L1 expression levels by immunohistochemistry have influenced the practitioners in a way to treat patients. There are also limitations for immunohistochemistry staining, distinct antibodies have unique sensitizers. Additionally, the threshold value of PD-L1 staining is still a discussion between pathologists and oncologists. Hence there is a great challenge for researchers, clinicians, and patients, antibody clones have influenced the distinctive epitope of the PD-L1 molecule with unique scoring systems based on the assays [44].

In inclusion to PD-L1, a few other immunosuppressive molecules such as TGFB1 and NKG2D ligands were also augmented in TD-EVs and were capable to prompt T-cell suppression [45]. NKG2D is a receptor that is activated by NK cells and a few subgroups of T cells and acts as a prime recognition receptor for the detection and elimination of cancer cells. These are stress-influenced self-proteins, which are released as soluble molecules through protease-mediated cleavage. An excretion of NKG2D ligands is regarded to maintain their expression levels related to the immune evasion mechanism occupied by tumor cells to avoid NKG2D-mediated immune observation [46]. It has been observed that TD-EVs from ovarian cancer and melanoma expressed NKG2D ligands and intercept activation of cytotoxic NK cells [47]. FASL and TRAIL expression on TD-EVs persuades apoptosis in dendritic cells (DCs) leading to immunosuppression and stimulating the progression of tumors [48]. TD-EVs on FASL terminated an antigen-specific effector T cells. There are few other immunosuppressive proteins like COX2, CD39/CD73, PDL1, FASL, TGFβ, CTLA4, TRAIL, etc., are appeared to relate to the TD-EVs [49].

For a better immunotherapy effect, the immunosuppressive part of TD-EVs would be impeded, to initiate the immune system. For this case, DCs are the foremost step in the immunity cycle for terminating the cancer cells via T-cell activation [50]. The surface membranes are easily attracted with the immune cells, in which the dendritic cells procured extracellular vesicles can likely be manipulated to behave as anti-cancer vaccines thereby leading to novel immunotherapy to fight against cancer. The surface molecules of the TD-EVs would be guided to deliver cancer treatment. Hence, the tumor-associated antigens, immunogenic peptides, and heat shock proteins would bring about cancer treatment shortly [51].
