**1.1. Heat shock proteins (HSPs)**

HSPs are a class of chaperone proteins ubiquitously expressed in the cells of both prokaryotic and eukaryotic organisms. HSPs have been traditionally named and subdivided into six groups or families based on their molecular weight, namely, the small HSPs (which include HSP27), HSP40, HSP60, HSP70, HSP90 and HSP100 family. However, more recently, a new nomenclature and classification system based on the naming issued by the Human Genome Organisation (HUGO) Gene Nomenclature Committee (HGNC) has been proposed for classifying human HSPs into the following groups: HSPA (HSP70), HSPB (small HSPs including HSP27), HSPC (HSP90), HSPD/HSPE (HSP60/HSP10), HSPH (HSP110) and DnaJ (HSP40) [1]. Each of these families has members that are constitutively expressed and others that are inducible upon stress.

Under normal physiological conditions, constitutive HSPs fulfil important regulatory roles in a wide range of cellular processes including the synthesis, folding, translocation, assembly and in some cases activation of the proteins they interact with. On the other hand, after an episode of cellular stress, inducible HSPs help to refold and prevent aggregation of misfolded proteins, as well as assist in the proteasomal degradation of misfolded proteins which cannot be recovered. Moreover, HSPs can block apoptotic signalling and increase tolerance to subsequent insults [2].

However, it is now starting to emerge that during stress, the role of HSPs goes beyond what is expected to be their intracellular chaperoning functions for recovery from multiple stress conditions. Despite HSPs acting predominantly intracellularly, they have also been found expressed in the cell plasma membrane and in the extracellular space. Numerous HSPs have been reported to be present in the extracellular space and general circulation, activating a range of signalling pathways depending on the effector cell type or target organ. The role of such extracellular HSPs appears to be that of a systemic warning system of stressful events or chronic conditions, acting by priming the body, of which the immune system is a major effector, in order to prepare for and counteract the spread of the stress insult. Extracellular HSPs thus seem to act as a form of intercellular communication system during stress conditions, particularly those responses linked to oxidative stress, immunity or inflammation [3].

### **1.2. The presence of HSPs outside cells**

When HSPs are present outside cells, they can be found as free proteins in solution or forming part of EVs. EVs can be of various types, with distinct structural and biochemical properties 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 apoptotic vesicles produced upon cell death [4, 5].

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

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

HSPs are a class of chaperone proteins ubiquitously expressed in the cells of both prokaryotic and eukaryotic organisms. HSPs have been traditionally named and subdivided into six groups or families based on their molecular weight, namely, the small HSPs (which include HSP27), HSP40, HSP60, HSP70, HSP90 and HSP100 family. However, more recently, a new nomenclature and classification system based on the naming issued by the Human Genome Organisation (HUGO) Gene Nomenclature Committee (HGNC) has been proposed for classifying human HSPs into the following groups: HSPA (HSP70), HSPB (small HSPs including HSP27), HSPC (HSP90), HSPD/HSPE (HSP60/HSP10), HSPH (HSP110) and DnaJ (HSP40) [1]. Each of these families has members that are constitutively expressed and others that are

Under normal physiological conditions, constitutive HSPs fulfil important regulatory roles in a wide range of cellular processes including the synthesis, folding, translocation, assembly and in some cases activation of the proteins they interact with. On the other hand, after an episode of cellular stress, inducible HSPs help to refold and prevent aggregation of misfolded proteins, as well as assist in the proteasomal degradation of misfolded proteins which cannot be recovered. Moreover, HSPs can block apoptotic signalling and increase tolerance to

However, it is now starting to emerge that during stress, the role of HSPs goes beyond what is expected to be their intracellular chaperoning functions for recovery from multiple stress conditions. Despite HSPs acting predominantly intracellularly, they have also been found expressed in the cell plasma membrane and in the extracellular space. Numerous HSPs have been reported to be present in the extracellular space and general circulation, activating a range of signalling pathways depending on the effector cell type or target organ. The role of such extracellular HSPs appears to be that of a systemic warning system of stressful events or chronic conditions, acting by priming the body, of which the immune system is a major effector, in order to prepare for and counteract the spread of the stress insult. Extracellular HSPs thus seem to act as a form of intercellular communication system during stress conditions,

particularly those responses linked to oxidative stress, immunity or inflammation [3].

When HSPs are present outside cells, they can be found as free proteins in solution or forming part of EVs. EVs can be of various types, with distinct structural and biochemical properties

cells (CTCs)).

102 Liquid Biopsy

signalling pathways during stress conditions.

**1.1. Heat shock proteins (HSPs)**

inducible upon stress.

subsequent insults [2].

**1.2. The presence of HSPs outside cells**

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 and bringing about metastasis [7, 8].

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 insult prior to its spread [3].

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

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 the healthy condition.
