**1. Introduction**

Liquid biopsies may contain a wide variety of biomolecules including DNA, RNA, proteins and metabolites. When considering the presentation of the numerous proteins within a liquid

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

biopsy, these can be free in the plasma, encapsulated within or on the surface of extracellular vesicles (EVs) or still inside cells within the biopsy (such as in the case of circulating tumour cells (CTCs)).

as well as intracellular site of origin. These include large microvesicles (up to 1500 nm) that are heterogeneous in shape and produced from the plasma membrane, small (50–100 nm) and more uniformly shaped exosomes released from endosomes via the endocytic pathway and

The Diagnostic and Prognostic Application of Heat Shock Proteins and their Post-Translational…

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EVs are released by almost all cell types, both healthy and diseased (including tumour cells). Such vesicles carry a wide range of biologically active molecules including growth factors, cytokines, mRNAs and microRNAs, extracellular matrix constituents and also proteins [6]. The protein fraction consists of cytosolic or plasma membrane components, either inside or on the surface. Their molecular contents have been shown to mediate intercellular communication in a variety of cellular processes, in both normal and pathological conditions, with the transfer of such biomolecules altering the function of the target cells. In the context of cancer, for example, EVs can modulate both the tumour microenvironment and cells and tissues which are located at a distance, affecting the immunity in the area, promoting angiogenesis

EVs are also released by cells in response to being exposed to a stressor or as a result of chronic cellular stress. Such EVs contain particular molecules, including HSPs, whose expression level is directly linked to or induced by the stress insult. Upon reaching their effector cells, and especially when interacting with cells of the immune system, some EV components act as signalling molecules, activating a response in the effector cells which pre-empts the stress

Proteomic studies have shown that EVs from serum, saliva, milk or plural effusions contain HSP27, HSP60, HSP70 and HSP90 [9–16] at high concentrations, with the ability to synergise with other encapsulated factors [3]. The delivery of HSPs in EVs provides a much stronger signal to effector cells as exemplified by EVs containing HSP70 producing a 250-fold higher

The HSPs encapsulated within or presented on the surface of such EVs, together with changing levels in free HSPs, can thus be valuable disease biomarkers for early detection, diagnosis and therapy selection. However, in order to access them, these proteins need to be purified from the body fluids of patients, characterised, quantified and compared to what is known in

When cells are exposed to a stressor, which includes but is not limited to heat shock, osmotic stress, exposure to heavy metals, hypoxia, ischemia or pathogens, these release signalling molecules in order to alert the rest of the system that a stressful condition is being experienced in some part of the organism and which might potentially lead to a situation of systemic damage. Among the stress signals which can be released by cells in response to an incidence of cellular stress are HSPs and other components of the chaperone (**Figure 1**). It is worth noting that most HSPs lack the consensus signal required for secretion via the classical endoplasmic reticulum (ER)-Golgi pathway [3, 18]. So far, it appears that the secretion of HSPs is achieved via a number of alternative pathways; however, these are still not well defined. Presently, the

activation of macrophages than an equal concentration of HSP70 in solution [17].

**2. Secretion and uptake of extracellular heat shock proteins**

HSP release mechanisms identified are (but might not be limited to) processes via:

apoptotic vesicles produced upon cell death [4, 5].

and bringing about metastasis [7, 8].

insult prior to its spread [3].

the healthy condition.

One class of proteins garnering particular interest as part of liquid biopsies are extracellular heat shock proteins (HSPs), and their post-translational modifications (PTMs), mainly because they should not be present in body fluids at the concentrations observed due to their lack of an export sequence and also as a result of the growing evidence supporting the notion that these proteins can mediate intercellular crosstalk and act as messengers that activate signalling pathways during stress conditions.
