Circulating biomarkers of cell death

Journal Pre-proofs Review Circulating Biomarkers of Cell Death Kerstin Wimmer, Monika Sachet, Rudolf Oehler PII: DOI: Reference:

S0009-8981(19)32067-4 https://doi.org/10.1016/j.cca.2019.10.003 CCA 15860

To appear in:

Clinica Chimica Acta

Received Date: Revised Date: Accepted Date:

10 April 2019 2 October 2019 3 October 2019

Please cite this article as: K. Wimmer, M. Sachet, R. Oehler, Circulating Biomarkers of Cell Death, Clinica Chimica Acta (2019), doi: https://doi.org/10.1016/j.cca.2019.10.003

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Circulating Biomarkers of Cell Death Authors: Kerstin Wimmer, Monika Sachet, and Rudolf Oehler# Authors’ affiliations: Department of Surgery and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria #Corresponding authors: Rudolf Oehler ([email protected]), Dept. of Surgery, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. Phone +43 1 40400 73513 Key words: biomarker, cell death, apoptosis, necrosis, necroptosis Conflict of interest: The authors declare no potential conflicts of interest.

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ABSTRACT Numerous disease states are associated with cell death. For many decades, apoptosis and accidental necrosis have been assumed to be the two ways how a cell can die. The recent discovery of additional cell death processes such as necroptosis, ferroptosis or pyroptosis revealed a complex interplay between cell death mechanisms and diseases. Depending on the particular cell death pathway, cells secrete distinct molecular patterns, which differ between cell death types. This review focusses on released molecules, detectable in the blood flow, and their potential role as circulating biomarkers of cell death. We elucidate the molecular background of different biomarkers and give an overview on their correlation with disease stage, therapy response and prognosis in patients.

GRAPHICAL ABSTRACT

HIGHLIGHTS: 

Regulated cell death such as apoptosis leads to a modulation of intracellular components

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They are eventually released from the dying cell and leak into the blood stream

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Cell death specific protein patterns can be detected in peripheral blood

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This review reports on currently used circulating biomarkers of cell death, describes their molecular background and summarizes their application in clinical studies.

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MAIN TEXT 1. Introduction Cell death mechanisms play a crucial role in maintaining hemostasis. These important mechanisms eliminate cells that are senescent, infected, damaged or carry mutations leading to cell cycle defects [1]. Deficient as well as excessive cell death mechanisms are involved in the development of various diseases. For example, chronic accumulation of dead cell debris is considered to initiate autoimmunity as in the case of systemic lupus erythematosus (SLE) [2]. Further, excessive apoptotic cell death is involved in neurodegenerative disorders [3] and immunodeficiency [4]. In the field of cancer, chemotherapeutic drugs aim to eliminate tumor cells by cancer cell specific cytotoxicity. Remarkably, some types of chemotherapy-induced cell death exceed the pure antineoplastic effect. Anthracyclines, a widely used class of chemotherapeutic agents, are known to induce an immunogenic cell death (ICD). Anthracycline-treated tumor cells release immunogenic damage associated molecular patterns (DAMPs) such as HMGB1 or calreticulin which stimulate antigen presenting cells and promote a T-cell response against tumor cells [5]. The knowledge about the pivotal role of cell death in different diseases prompted many researchers to explore whether the degree of cell death can be used as a biomarker to monitor the progression of the disease, to predict the response to therapy, or to estimate patient's prognosis. Most studies analyze directly the affected tissue using immunological or enzymatic methods. For example, they visualize apoptotic cells in the tissue by immunohistochemistry using antibodies against active caspase 3 or caspase-cleaved proteins [6], [7]. Alternatively, they identified dying cells by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), an in-situ detection of fragmented DNA [7]. The analysis of tissue sections provides accurate information about which cell type is dying and how does the surrounding tissue react to this process. Unfortunately, the availability of tissue specimens is often limited and consequently clinicians give precedence to clinical purposes. A further important issue in cell death research is the correct timing and precise assay design. The here presented review focusses on cell death related molecules released by dying cells and reports about

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the use of these substances, detectable in the peripheral blood, as circulating biomarkers of cell death. It describes their molecular background and gives an overview on their applicability in clinical practice. In order to assess the clinical significance of these markers it is necessary to understand the molecular processes of cell death in some detail.

2. Cell death processes All cells have a limited time span, which can range from a few days as in the case of neutrophils [8] to many decades as for neurons [9]. Consequently, billions of cells die every day and are quickly removed by neighboring phagocytes [10], [11]. The efficiency of this clearance is demonstrated by the fact that dying cells are hardly detectable in healthy tissue by conventional histology [12]. Pathological conditions such as infection, inflammation, intoxication or extreme environmental situations can lead to a strong local increase of cell death. Harsh noxious stimuli can induce massive cell damage which results in an immediate disintegration of the cell. This form of cell death is termed "accidental necrosis" or "primary necrosis" and is characterized by a sudden release of intracellular molecules which act outside of the cell as DAMPs and initiate a strong pro-inflammatory immune response [13]. In contrast, milder but still not repairable cell damage induces a regulated form of cell death which delays the release of intracellular content and dampens the inflammatory response. For decades, apoptosis was assumed to be the only form of regulated cell death, ensuring a controlled dismantling of cell structures and a consecutive disposal of the dying cell without disturbance of neighboring cells and structures [14]. Ongoing investigations revealed evidence for further forms of regulated cell death mechanisms such as necroptosis, ferroptosis, NETosis, pyroptosis, entosis, parthanatos and mitotic catastrophe. It has to be expected that this list will grow in the next years. A recent consensus paper, written by Lorenzo Galluzzi and more than 160 experts in the field of cell death research, provides a widely accepted nomenclature of different forms of regulated cell death [15]. The molecular background and pathological features of these different forms have been extensively summarized recently elsewhere [15], [16], [17], [18], [19]. In case of apoptosis, the cell

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membrane integrity is preserved over nearly a day [20]. During this time period, effector caspases degrade intracellular structures and organelles which are then released in small extracellular vesicles. This leads to a shrinkage of the remaining cell remnant, facilitating the uptake by phagocytes before cell membrane disruption [21]. Thus, apoptotic cells can be cleared without any induction of inflammation. In contrast, most other forms of regulated cell death result in an earlier loss of membrane integrity and are associated with a release of pro-inflammatory molecules. These forms are therefore often summarized under the term "regulated necrosis", indicating a limited release of modified intracellular molecules and a reduced inflammatory response as compared to accidental necrosis. For example, necroptotic cells retain their membrane intact for a few hours [22]. During this time, necroptotic cells undergo a distinct processing of intracellular molecules, which differs from apoptosis. The type of cell death, which is induced as a reaction onto a particular stimulus, depends on the cell death signal, the type of tissue, the developmental stage of the tissue, and the physiologic milieu. Consequently, the same inducing trigger may evoke simultaneously different types of cell death in neighboring cells of the same tissue. Dead cells are usually only detectable when they accumulate in a tissue whenever the death rate exceeds the clearance rate. If dying cells are not cleared in time, cell debris with a morphological aspect of necrosis will accumulate. The possibilities to identify the underlying type of cell death by classical histology are limited. The term ‘‘necrosis’’ was therefore over a long time used by pathologists in accordance to the “Nomenclature of Cell Death Committee” of the Society of Toxicologic Pathologists to describe any morphological findings of cell death in histological sections, regardless of the underlying cell death pathway [23]. Significant cell death consists usually of clusters of many necrotic cell remnants which represent the very late form of cell death of different and potentially mixed molecular background. Such clusters may either preserve the outline of the tissue architecture (coagulative necrosis) or degrade completely to a liquid viscous mass (liquefactive necrosis). Because of the complete disintegration of cell membrane, the cell content is released into the microenvironment and might find its way into the circulation. Accidental necrotic cells release exclusively unmodified proteins. In contrast, all forms of regulated cell death are associated with page 5 of 48

post-translational modifications of proteins such as proteolytic cleavage or phosphorylation of specific molecules. Apoptosis, for example, mediates the activation of the effector caspases 3, 6 and 7 by proteolytic cleavage. The resulting subunits form a proteolytically active heterodimer which cleaves its substrate at a distinct amino acid sequence of Asp-Glu-Val-Asp, the so-called DEVD motive [24]. More than 300 substrates of activated effector caspases have been identified so far [25]. Thus, apoptosis provides many different potential biomarker candidates. Products of necroptosis, in contrast, are less explored yet and therefore only a few biomarkers are currently available (e.g. mixed lineage kinase domain like pseudokinase, MLKL) [17]. It has to be expected that the identification of promising necroptosis markers will increase in the next years. However, necroptosis and mostly all other forms of regulated necrosis are much quicker and less complex than apoptosis. Thus, the number of potential biomarkers for these forms of cell death might remain limited. Figure 1 gives an overview on molecular structures which are released from dying cells and have been used to detect cell death in blood samples. The details are discussed below. Although most of these circulating biomarkers are related to apoptosis, we will also discuss very recent studies on circulating biomarkers of necroptosis.

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3. Circulating biomarker for apoptosis 3.1 Caspase 3 Caspase-3 is a cysteine protease that is - if activated - an important effector of apoptosis as it is capable of disassembling the cell. It is therefore a hallmark of apoptosis. In various extracellular fluids the protease retains its tetrapeptide sequence DEVD cleaving activity over a long time [26]. This proteolytic reaction can be also catabolized by other caspases, which are all involved in apoptosis (i.e. caspases 2, 6, 7, 8 and 9, 10). Nevertheless, a circulating DEVD cleaving activity is usually termed as "caspase 3/7 activity" because these effector caspases are the most abundant [27]. It has been proposed as a specific circulating biomarker reflecting apoptosis in the tissue [26]. For example, treatment of colorectal cancer with 5FU induces caspase 3 activation in cancer tissue [28]. Accordingly, colorectal cancer patients show an increase in serum caspases 3/7 activity after administration of 5FU-based chemotherapy [29]. Breast cancer patients show an elevated serum DEVD-cleaving activity that correlates with tumor stage [30]. Several studies confirmed an increase in circulating caspase 3/7 activity in critically ill patients. For example, cerebrospinal liquor samples of head trauma patients contain an about three-fold higher DEVD-cleaving activity than healthy controls [26]. These data show that caspase 3/7 activity is a promising circulating biomarker for apoptotic cell death. Although enzymatic assays are usually complex to perform, there are ready-touse kits available that allow an easy detection. Most authors used the Promega Caspase-Glo 3/7 assay (Promega Corp., Madison, WI, USA). It applies a proluminescent caspase substrate containing the DEVD motive. Circulating active caspase cleaves this substrate resulting in a luminescent signal. Some recent studies quantitated the total caspase 3 protein by ELISA instead of its enzymatic activity. It was observed that serum caspase 3 levels increase in some disease conditions, e.g. after aneurysmal subarachnoid hemorrhage [31], intracerebral hemorrhage [32], severe traumatic brain injury [33], and severe sepsis [34]. The ELISA kits do not differentiate between active and inactive caspase 3. The excretion mechanism, which releases caspase 3 protein is released into the circulation

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is unclear. It may be related to necrotic cell death but further studies are required to proof this assumption.

3.2 Caspase cleaved cytokeratin 18 During apoptosis the effector caspases cleave hundreds of different proteins including cytokeratin 18 (CK18). CK18 is highly expressed in various tissues such as liver, intestine, lung, kidney, bone marrow, endocrine tissues, and many solid tumors of epithelial origin [35]. However, it is nearly absent in brain, muscle, skin, and immune cells. CK18 is cleaved by effector caspases at two distinct sites (Asp238 and Asp396) [36]. Cleavage after Asp396 leads to exposure of the M30 neo-epitope [37]. Antibodies against M30 are used to detect caspase cleaved CK18 (ccCK18) in tissue sections. The specificity of this antibody to detect apoptotic cells has been characterized in the intestine [6]. The intestinal epithelium is constantly renewed by stem cells located at the bottom of the crypt. During maturation they move towards the villus tip where they become senescent and die, exhibiting typical morphological characteristics of apoptosis (apoptotic bodies, nuclear condensation, cytoplasmic shrinkage and membrane blebbing). Koornstra and coworkers showed that the M30 antibody binds specifically to these apoptotic cells [6]. Based on the number of positive cells, the authors calculated an apoptosis index describing the percentage of apoptotic cells. Regular mucosa adjacent to colorectal tumor tissue, showed a two-fold higher apoptosis index than distant regular mucosa. The cancer tissue itself showed a 10-fold higher index. It is commonly associated with hollow spheroid nests, characterized by a remarkable spheroidal layer of cancer cells filled with disintegrated dead cells. Using the M30 antibody, Tamura and co-workers showed that these dead cells underwent an apoptotic cell death pathway before they became secondary necrotic [38]. Their presence is an independent risk factor for metastases and shorter survival. Molecular degradation products of these areas of massive apoptosis are released eventually to the circulation. Accordingly, colorectal cancer patients show a higher ccCK18 in their blood flow as compared to healthy controls [39]. The circulating ccCK18 was quantitated by a specific ELISA which combines the M30 antibody with an antibody against the antigen M5 on the same cleavage product [37]. This ELISA is usually compared page 8 of 48

with an M65 ELISA which quantitates total circulating CK18 (caspase-cleaved and non-cleaved) using antibodies against the antigens M6 and M5 (see also Figure 1). A positive signal in M30 and M65 indicates apoptosis, whereas an exclusive positive signal in M65 points to necrosis. The group of Carolyn Dive published a detailed analysis on the stability of M30 and M65 antigens in blood samples [40]. They showed that although it can be used to analyze serum as well as plasma samples, the variation between duplicates is lower in serum. In order to avoid artificial cell death during the time between acquisition and processing, samples should be placed immediately on ice. Most importantly, prolonged storage at -80°C leads to an unspecific increase of the M65 signal. Micha and co-workers proofed the specificity of circulating ccCK18 in an animal model of small cell lung cancer [41]. Circulating ccCK18 increased in response to treatment with ABT-737, a selective inhibitor of Bcl2 that plays an important role in cell curvival, and correlatet with an increase of the number of active caspase-3 cells in the tumor tissue. Many studies used the M30/M65 ELISA combination to investigate samples from cancer patients. For example, blood samples from small cell lung cancer patients showed elevated levels of M30 and M65 [42]. The M30 signal correlated with the percentage of circulating apoptotic tumor cells. Both levels were prognostic for patient survival. M30 further increased 48 hours after initiation of chemotherapy, whereas the number of circulating tumor cells decreased. The authors concluded that the therapy induces apoptotic tumor cell death resulting in a reduced number of malign cells and a concomitant increase of circulating tumor cell debris. Patients with untreated malign endometrial cancer showed barely detectable peripheral blood M30 levels, similar as in patients with benign endometrial conditions [37]. However, the local pelvic blood levels were clearly higher in the malign than in the benign setting. This indicates that the local increase in soluble ccCK18 was diluted in the circulation and became undetectable. The combination of M30 and M65 ELISAs is an easily performed test. A sequential analysis of cell death biomarkers during an anti-neoplastic treatment period of cancer patients might be a promising approach to determine therapy response in a longitudinal setting. However, it has to be considered that not all cancer types express CK18 and its expression level vary considerably between patients within a specific cancer type [35]. It is for example absent in the great majority of skin cancers as well page 9 of 48

as in head and neck cancers, whereas most lung cancers are strongly CK18 positive. However, in about one out of four lung cancer patients very low CK18 expression are found. Thus, a low M30 signal in the peripheral blood does not exclude ongoing apoptosis. The situation in non-malignant tissues is much clearer. For example, CK18 is a major intermediate filament protein in the liver. The M30/M65 assay is therefore used as a non-invasive blood marker to monitor hepatocytic cell death (summarized in Eguchi et al. [43]). The quantitative assay was applied to determine hepatotoxic effect of drug compounds but also to detect disease-associated hepatocytic cell death. A recent meta-analysis confirmed that the quantification of ccCK18 fragments in plasma samples exhibits a high sensitivity and specificity in the diagnosis of non-alcoholic steatohepatitis (NASH) [43]. Despite the fact that the M30/M65 ELISA is restricted to CK18 expressing tissue, it is a very frequently used circulating biomarker of cell death. However, to confirm the tissue of origin of circulating ccCK18 it is advisable to analyze selected tissue samples for apoptosis either by using antibodies against M30 or by other appropriate histological methods such as a TUNEL assay.

3.3 Nucleosomes and circulating DNA Another target of activated effector caspases is the inhibitor of caspase activated DNase (iCAT). A degradation of iCAT activates the DNase and leads to a cleavage of chromosomal DNA between nucleosomes. This process is termed “DNA laddering” because of the separation pattern of the resulting fragmented DNA in an agarose gel [44], [45]. When apoptotic cells progress towards secondary necrosis, they release nucleosomes into the supernatant. They are highly immunogenic and contribute for example to the development of autoimmune diseases such as systemic lupus erythematosus (SLE), where autoantibodies against double strand DNA play an important role [2]. Nucleosomes are stable in circulation with 7% decrease in nucleosome concentration per year in storage [46]. Elevated levels of circulating nucleosomes are frequently observed in different cancers and also in stroke, trauma, and sepsis as well as in autoimmune diseases (for review see [47]). Circulating nucleosomes are used as a non-invasive method for detecting cancer and monitoring response to treatment (for review see [48]). Chemotherapy induced a further increase of circulating page 10 of 48

nucleosomes in pancreatic cancer patients [49]. This increase was associated with poor outcome. In breast cancer patients, a similar trend between levels of circulating nucleosomes and tumor progression was observed [30]. However, circulating nucleosomes can be also observed in samples from healthy subjects limiting their diagnostic value. Furthermore, it has to be considered that the binding efficiency of DNA to nucleosomes is strongly related to the epigenetic methylation of the DNA itself as well as to post translational modification of the histone tail of the nucleosome [50]. Such DNA methylation and histone modification of nucleosomes are common in cancer tissue. Accordingly, the group of Stefan Holdenrieder could show that measuring the methylation marks on circulating nucleosomes improves the sensitivity and specificity for the detection of colorectal cancer as compared to total nucleosomes [51]. Similarly, Rahnier et al found that circulating nucleosomes with a colorectal cancer (CRC) specific epigenetic profile diagnosed stage I and II CRC with a higher high specificity and sensitivity as overall circulating nucleosomes [52]. It has been observed that circulating nucleosomes correlate in various diseases with the overall level of cell-free DNA (cfDNA) [30], [53]. Tumor-derived cfDNA (also termed "circulating tumor DNA" or ctDNA) has gained increasing importance in cancer research. Numerous studies confirmed that the analysis of tumor-derived cfDNA is a valuable tool for early diagnosis of various cancer types, to monitor tumor progression and response to treatment, and to detect metastatic disease early (reviewed in [54]). Interestingly, circulating cfDNA consists mainly of nucleosome-protected DNA that was released by apoptotic tumor cells into the blood stream [55]. Tumor derived cfDNA fragments have a length of a multiple of 166 basepairs (bp) after being enzymatically processed from an apoptotic cell. This number of basepairs corresponds with the length of DNA that is wrapped around a nucleosome (∼147 bp) plus a linking fragment (∼20 bp). Therefore, high levels of cfDNA with tumor-specific signatures indicate an elevated release of nucleosomes from apoptotic tumor cells. The research group of Michael Speicher proposed recently that the detection of 166bp mononucleosomal cfDNA can be used as an indicator for apoptosis without additional proof of circulating nucleosomal proteins [55]. Because the analysis of cfDNA applies a PCR amplification step it is by far

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more sensitive than a nucleosome detection using an ELISA. Thus, this new approach has the potential to bring the field of circulating biomarkers of apoptosis a big step further.

3.4 Extracellular vesicles Apoptosis is also characterized by a caspase-mediated contraction of sub-membranous actin-myosin cytoskeletal structures [56], [57]. This results in the formation of cellular protrusions, usually termed "apoptotic blebs", which are finally released from the cell remnant as apoptotic cell-derived extracellular vesicles (ApoEVs or aEV) [58]. This process, called blebbing, facilitates the fragmentation of the dying cell into smaller pieces, which can then be more easily cleared by neighboring phagocytes. Recent findings suggest that aEVs modulate also the immune response to apoptosis [10], [20], [58]. The greater part of aEVs consists of large apoptotic bodies with a diameter between 1 and 5 µm which often contain entire organelles. In addition, apoptotic cells release also apoptotic microvesicles (aMVs) with a size smaller than 1µm [58]. It has been increasingly recognized that also viable cells release extracellular vesicles (EV) in the process of normal functions [59]. Circulating EVs in healthy humans usually have a diameter of 0.03-1 µm and derive mainly from platelets, erythrocytes, monocytes, leukocytes and smooth muscle cells. In malignancy, EVs may also originate from viable cancer cells. They are implicated in trafficking molecules between cells and as such have an effect on physiologic function and serve as biomarkers for diseases (for review see [60]). Many studies found elevated levels of small EVs in the blood of patients with diverse pathological conditions (summarized in [59], [60]). Although EVs from healthy cells are distinctly smaller than the large apoptotic bodies, there still exists a size-overlap with aEVs. The differentiation between EVs and aMVs is further compounded since both expose phosphatidylserine (PS) on their surface due to the lack of flippase activity [61]. Thus, while a >1 µm circulating EV can be clearly attributed to apoptotic bodies, it remains extraordinarily difficult to assign a <1 µm EV to normal cell functions or to distinct cell death processes, unless the EV is caught in the act of its release by live imaging techniques [62]. We showed recently that bortezomib-based chemotherapy induces an increase of large circulating dead cell bodies in the circulation of patients with multiple myeloma within three page 12 of 48

days [63]. This accumulation of dead cells was associated with an impaired ability of monocytes to phagocytose apoptotic cells. The group of Johan van der Vlag found PS–positive EVs in patients with SLE that contained apoptosis-modified chromatin as well as endothelial markers on their surface [64]. These aEVs were able to induce a pro-inflammatory response of dendritic cells and primed neutrophils for NETosis. Notably, they were absent in blood samples of healthy controls. Intensive research during the last decade confirmed a connection between circulating EVs and venous thromboembolic events (VTE) in cancer. Various types of solid cancers, including prostate, head and neck, pancreatic, breast and renal cancer, express high levels of tissue factor (TF) [65], [66], [67]. VTE correlates with the number of TF positive EVs in the circulation of these patients. Furthermore, the presence of PS on EVs enhances the pro-coagulant activity of TF [68]. A recent animal model study showed that caspase inhibition reduces TF-driven coagulation [69]. This strongly suggests that TF-positive EVs are primarily aEVs, indicating a predominant role of apoptosis in VTE. Further corroboration for this assumption comes from the observation that chemotherapeutic agents increase the risk of VTE. Chemotherapy-induced apoptosis of tumor cells elicits an increased release of aEVs and a concomitant increase in blood-borne TF and pro-coagulative membrane surfaces, resulting in an enhanced risk of VTE. A similar relationship between apoptosis and TFinduced coagulation has been found in patients with cardiovascular disease [70]. PS–positive aEVs are produced in considerable amounts within the lipid core of atherosclerotic plaques. Additionally, a high expression of TF further contributes to the pro-coagulative properties of the lesion. Recent research showed that circulating EVs in patients with cardiovascular disease might also have completely different effects. Cardiac stem cell-derived EVs reduce apoptosis of adjacent cardiomyocytes [71]. Similar protective effect of tumor cell-derived EVs against apoptosis has been observed in prostate cancer [72]. They contained survivin and other inhibitors of apoptosis proteins (IAPs), which provided neighboring cells with a higher resistance against apoptosis inducer. Taken together, these results confirm that aEVs are released into the circulation, exerting different biological functions. However, their quantification is challenging and not standardized. Most studies differ in regard to EV preparation, staining and analysis. Therefore, the Society for Extracellular page 13 of 48

Vesicles defined the correct analytical requirements in a recently published consensus paper [62]. Still, the differentiation between aMVs and circulating small EVs remains challenging and an unambiguous assignment of circulating EVs to apoptosis is difficult. It can be summarized that circulating EVs can provide valuable data on disease state and progression but should be combined with other biomarkers of cell death.

4. Circulating biomarkers for necrosis and tissue damage As described above, accidental necrosis (or primary necrosis) results in an immediate unregulated release of unmodified intracellular molecules. In contrast, every form of regulated cell death leads to characteristic modifications of selected molecules. However, most molecules remain unchanged. If the dying cell is not cleared in time, the regulated cell death process proceeds until "secondary necrosis" and modified as well as many unmodified intracellular molecules are released [21]. Thus, unmodified molecules in the circulation could originate from primary necrosis or from secondary necrosis. Hence, biomarkers of "necrosis" or "tissue damage" comprise all dying cells with a ruptured plasma membrane, regardless of the initial cause of cell death. A classical biomarker is circulating enzymatic activity of lactate dehydrogenase (LDH). LDH is expressed in almost all cells and elevated activity in the peripheral blood indicates necrosis and tissue damage. The liver transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are examples of wellestablished tissue-specific biomarkers. The clinical value of these long-known biomarkers is summarized elsewhere [73], [74]. The present review focusses on novel markers of necrosis and tissue damage.

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4.1 High-mobility group box-1 protein The high-mobility group box-1 (HMGB1) protein is a conserved, abundant, non-histone nuclear protein expressed in almost all eukaryotic cells. HMGB1 binds to double-stranded DNA, singlestranded DNA, distorted DNA and nucleosomes. It stabilizes the structure of nucleosomes and induces DNA bending [2]. During apoptosis, HMGB1 gets tightly attached to hypoacetylated chromatin. Post-apoptotic secondary necrotic cells do not release free HMGB1 as it is tightly bound to nucleosomes [75]. All other form of cell death lead to a passive release of free HMGB1 resulting in an inflammatory response [76]. Furthermore, HMGB1 can be actively secreted by macrophages via an unconventional autophagy-based secretion pathway [77]. Interestingly, such secretion can be induced by different stimuli including extracellular HMGB1, which either originates directly from necrotic cells or indirectly from macrophages, activated in response to necrosis. HMGB1 has been classified recently as alarmin as well as an atypical chemokine [78]. Outside the cell, it binds with high affinity to several receptors on the surface of immune cells, including toll-like receptor (TLR) 2, TLR4 and RAGE (receptor for advanced glycation endproducts) [78]. The immunostimulatory activities of extracellular HMGB1 depend strongly on its redox state. Inflammatory-active HMGB1 binds to TLR4 and requires an intramolecular disulfide bond (Cys23 and Cys45) and a reduced Cys106. In contrast, the all-thiol conformation of HMGB1 forms a complex with CXCL12 and acts as a chemokine. Finally, the terminally oxidized sulfonyl-HMGB1 fully lacks any immunologic activity. Circulating HMGB1 is usually quantitated by an ELISA which cannot differentiate between the three oxidation forms of HMGB1. Nucleosomes bound HMGB1 is not detected as long as no denaturing processes are performed during sample preparation. Thus, the ELISA measures total free HMGB1, with its origin from either necrotic cells or activated macrophages. Daniel Antoine compared serum levels of total HMGB1 with those of total CK18 (M65) in acetaminophen overdose patients with and without liver injury [78]. Higher levels of both, HMGB1 and CK18, correlated with poor prognosis of these patients. Circulating HMGB1 was even found to be superior to serum alanine aminotransferase (ALT) levels in identifying acute liver injury [76]. Furthermore, HMGB1 is strongly elevated in septic patients [79]. Notably, HMGB1 seems to play an page 15 of 48

important role in autoimmunity. Patients with SLE showed higher levels of HMGB1-nucleosom complexes in their serum than healthy controls [2]. This increase was associated with antibodies against double-stranded DNA. The authors showed evidence that chronic release of HMGB1nucleosom complexes from post-apoptotic cells promote the formation of such autoantibodies. In case of a malignant disease, HMGB1 and its role were investigated in various studies. For example, patients with malignant pleural mesothelioma (MPM) show a higher level of HMGB1 in their peripheral blood than healthy controls [80]. Further, the level of HMGB1 correlated with prognosis MPM patients with higher serum HMGB1 had a worse prognosis. About ten years ago the research group of Zitvogel and Kroemer discovered that specific chemotherapeutic agents such as anthracyclines induce a release of HMGB1 which is associated with the development of an adaptive immune response against the tumor [81]. This effect was termed “immunogenic cell death (ICD)”. A recent consensus paper defined increased blood levels of HMGB1 as a hallmark of ICD [82]. The increase reflects an - at least partial – induction of a non-apoptotic cell death process. We could show recently that the level of circulating HMGB1 increases in breast cancer patients within three days after the initial dose of epirubicin/docetaxel – containing chemotherapy [83,84]. This increase correlated with a reduction of tumor volume at the end of chemotherapy and with the five-year overall survival of patients. Similarly, patients with colorectal cancer (CRC) showed an increased release of HMGB1 from tumor cells in response to neoadjuvant chemotherapy [85]. Again, this correlated with a better prognosis of the patients. Also radioembolization therapy of CRC patients results in an immediate increase of serum HMGB1 within 24 hours [86]. In contrast, in patients with advanced pancreatic cancer receiving chemotherapy a decrease in circulating HMGB1 was observed [49]. This decrease was more pronounced in patients with progressive disease and correlated with the overall survival. Interestingly, a similar chemotherapy-induced decrease was observed in the same samples for circulating nucleosomes while the levels of soluble RAGE (sRAGE) and DNAse increased. The authors proposed that this decrease is related to a reduced macrophage activity resulting in a diminished active release of HMGB1 by these cells.

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In summary, circulating total free HMGB1 is widely used for measuring the extent of disease-related or therapy-induced acute tissue damage. However, such conditions are always associated with inflammation and macrophage activation which may result in additional HMGB1 release. It is therefore strongly suggested to complement HMGB1 measurements with additional circulating or tissue biomarkers of cell death. In cancer patients, contradictory results regarding the correlation of of HMGB1 level increase or decrease during a certain therapy and prognosis where found. This seems to differ between cancer types and administered chemotherapy. Ongoing research might be useful.

4.2 Extracellular microRNA MicroRNA (miRNA) is a class of 19-24 nucleotides long non-coding RNA molecules that regulate gene expression at post-transcriptional levels. MicroRNAs are formed in the nucleus, processed on their way to the cytoplasm and finally loaded onto an Argonaute (AGO) protein. Such miRNA-AGO complexes can bind to complementary 3’-untranslated regions of target mRNAs, decreasing target stability and translation efficiency. According to the miRBase database (http://www.mirbase.org), about 1900 different human miRNAs have been identified so far. Their expression is highly tissuespecific. A recent systematic analysis including 46 primary cell types and 26 tissues showed that about every third miRNA is present in only a single class of cell whereas merely 1.9% are present in every cell type [87]. About a decade ago it was discovered that miRNAs are also released into the blood flow [88]. These extracellular miRNAs (ex-miRNA) are vesicle free miRNA-AGO ribonucleoprotein particles (RNP) [89]. In contrast to free RNAs, which are rapidly degraded by RNases, RNPs are stable over a long time in human plasma. It has been shown that ex-miRNA can mediate short-range communication between different cells. For example, activated platelets release functional ex-miRNA-223 which is taken up by endothelial cells and regulates the ICAM1 expression of these cells [90]. However, the ability of ex-miRNAs to mediate distant cell-cell communication is under discussion. The concentration of total ex-miRNA in the blood is with 10-10 mol/l far below that concentration which is required in in vitro experiments to modulate target cells [89]. Therefore it has page 17 of 48

been proposed that ex-miRNAs in the peripheral blood are merely byproducts of cell activity and cell death without any particular function [89]. Because miRNA analysis include a PCR amplification step, it is possible to detect very low concentrations of ex-miRNA in plasma and serum. This prompted Kai Wang and colleagues to use hepatocyte-derived ex-miRNAs for a sensitive monitoring of druginduced liver injury [91]. They could show that the plasma concentrations of the most abundant liver miRNA species (mir-122 and mir-192) increase in a dose- and exposure duration-dependent manner. They observed that the increase of these ex-miRNAs was more sensitive than the classical liver toxicity parameter ALT. The concept to use ex-miRNA as a tissue-specific and sensitive cytotoxicity marker was confirmed in other tissues as well. A single administration of neurotoxic trimethyltin to rats induced an elevation of circulating mir-9* and mir-384 levels [92]. This increase correlated with neuronal apoptosis. Similarly, ex-miRNA increase also in diseases which induce necrosis and tissues damage. For example, the circulating levels of mir-21, which is highly expressed in the myocardium, was found to reflect left ventricular fibrosis in aortic stenosis patients [93]. A loss of pancreatic beta cells is commonly associated with the development of diabetes type I and II. Farr et al. implemented the idea of using islet-specific miRNA as a biomarker to diagnose as well as to monitor maintenance and regeneration of pancreatic beta cells [94]. Patients with non-alcoholic fatty liver disease (NAFLD) showed elevated ex-miRNA mir-21, mir-34a, mir-122, and mir-451 [95]. The serum concentrations correlated with the severity of liver steatosis. Note that it remains unclear whether the increase of ex-miRNA is exclusively the result of an unspecific release from dying hepatocytes or derives (at least partially) also from neighboring liver cells which became activated during this process. Accordingly, Lambrecht and coworker showed that the elevated plasma levels of miRNA mir-122 and mir-200b in early stage chronic hepatitis derive from hepatic stellate cells rather than dying hepatocytes [96]. These data show that although ex-miRNA can be very sensitive biomarker for necrosis and tissue damage they can also derive from living cells. Thus, an additional marker is needed to proof cell death.

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5. Circulating biomarkers for necroptosis 5.1 RIPK3-MLKL-pathway The most intensively investigated subroutine of regulated necrosis is necroptosis [97], [98], [99]. This form of cell death leads to a plasma membrane rupture without prior cell shrinkage. The membrane disintegration is facilitated by the phosphorylated form of mixed lineage kinase domain-like pseudokinase (MLKL; detailed reviewed in [16,17,100]). During the induction phase of necroptosis MLKL forms a complex with receptor-interacting serine-threonine kinases 1 and 3 (RIPK1 and RIPK3). RIPK1 is involved in the regulation of both, necroptosis and apoptosis through interaction with caspase-8. If the caspase-8 pathway is blocked, RIPK1 acts as a regulator of RIPK3-depended MLKL phosphorylation [101]. The presence of RIPK1, RIPK3, and MLKL is essential for necroptosis [102]. Several studies quantitated the expression levels of RIPK1, RIPK3, or MLKL in cancer patient samples and uncovered strong differences between cancer types. Immunohistochemical studies in colon cancer [103] and ovarian cancer [104] showed that tumor cells contain high amounts of MLKL protein whereas it was nearly undetectable in the adjacent normal tissue. In contrast, normal gastric tissues express high levels of MLKL mRNA as measured by quantitative rtPCR [105]. The level in gastric tumor cells is reduced. Similarly, Western blotting experiments revealed a high expression of RIPK3 in normal breast tissue while it is reduced in tumor cells [106]. In spite of these MLKL variations between different tissues, most studies observed a similar relation between tumor MLKL expression and prognosis. Low tumor MLKL assessed by immunohistochemistry was associated with decreased overall survival in colon cancer [103], ovarian cancer [104], pancreatic adenocarcinoma [107] and gastric cancer [105]. Accordingly, a recent meta-analysis including six eligible studies with 613 patients with different solid cancers showed that decreased expression level of MLKL was significantly associated with poor overall survival (pooled HR 0.26) and event-free survival (pooled HR 0.45) [108]. These results fit well with the concept of the pro-inflammatory properties of necroptotic cell death which is supposed to boost the anti-tumor immune response. Thus, although the quantitation of total MLKL by IHC is not sufficient enough to prove necroptotic cell death, it

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represents an interesting biomarker of prognostic value in solid cancers. Future studies on the phosphorylation status of cancer cell-derived MLKL might help to gain a greater insight into the role of necroptosis in cancer. Necroptosis also seems to play a role in non-cancerous diseases. The analysis of spinal cord samples of amyotrophic lateral sclerosis (ALS) patients revealed an increased expression of RIPK1, MLKL, pMLKL, but no difference in RIPK3 when compared to control tissue [109]. Another study analyzed ileum and colon samples of children with inflammatory bowel diseases (IBD) by Western blotting [110]. The experiments showed that RIP3 and MLKL were higher in inflamed than in the normal tissues. Similar Western blotting analyses of left ventricular samples of patients with end-stage heart failure showed a higher expression of MLKL and its upstream molecules RIPK1 and pRIPK3 as compared to healthy controls [111]. Necroptosis results eventually in a release of intracellular components. Based on this observation several recent studies investigated the concentration of the necroptosis related proteins MLKL and RIPK3 in the peripheral blood using specific ELISAs. Critically ill patients showed at the day of admission to the ICU similar serum MLKL levels as healthy controls [112]. However, the level changed over time. An elevated level of MLKL after three days of ICU treatment was predictive for patients' mortality. Only a few studies investigated the levels of these necroptosis markers in the circulation and not in the tissue. The serum level of RIPK3 showed a similar pattern in patients with acute kidney injury [113]. It increased within 48 hours. In case of resuscitation, the degree of increase correlated with the severity of the disease and with the number of applied red blood cell units. Critically ill patients requiring mechanical ventilation may develop ventilator-induced lung injury (VIL), which is associated with alveolar cell death. Patients with VIL showed higher plasma levels of RIPK3 than those without, indicating a contribution of necroptosis [114]. Interestingly, the level of MLKL did not significantly differ between these two groups. Taken together, many studies on necroptosis-related proteins quantify their overall concentration. Only a few determined their phosphorylation. This might be explained by the fact that a proper antibody against pMLKL has only recently become available. However, only the phosphorylation status would be a valid proof for an ongoing necroptosis. Nevertheless, the quantification of MLKL, page 20 of 48

RIPK1 or RIPK3 in tissue samples revealed that their expression level correlates with patients’ prognosis in many diseases. Till date, there are only few and very recent studies investigating these proteins in plasma or serum samples. Considering the huge interest in necroptosis, we expect that this number will increase in the coming years.

6. Laboratory aspects and timing of biomarker research As shown in Table 1, the described circulating biomarkers include enzymatic activity, proteins, nucleotides, heteromeric complexes, and subcellular vesicles. Thus, completely different analytical methods for quantification are required which may lead to divergent results. In case of biomarkers for apoptosis, the quantification of caspases and their enzymatic activity differ greatly. Caspases, which are proteins, can be measured with ELISA technique by using plasma or serum. Nevertheless, an accurate conclusion to the extent of apoptosis cannot be drawn since a differentiation between activated and total caspase amount is not possible. Therefore, the enzymatic activity testing adds additional information. Lee et al. described the detection of DEVDase in apoptotic cells by using a fluorogenic substrate, (z-DEVD)2-cresyl violet [115]. This substance is a cell permeant, fluorogenic, caspase substrate that detects caspase-3 and caspase-7 up-regulation in apoptotic cells, in this case in a cell culture model. The optimal concentration of the fluorogenic substrate, however, depended on the cell type as well as on timing and temperature. For the detection of caspase cleaved cytokeratin 18 an ELISA technique is recommended. Plasma as well as serum samples can be used for the quantitative testing of this protein. A limitation is that ccCK 18 quantification is restricted to those target tissues that are CK18 expressing it selves. For detailed information about the combined use of additional ELISAs to detect cleavage products of ccCK18 please read the section 3.2. Nucleosomes can be detected in peripheral blood, e.g. plasma, by using ELISA technology. Nucleosomes are considerably stable. Nevertheless storage time must be taken into account since an annual loss of 7% has to be expected in stored samples at -70°C [46].

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To determine the concentration of cfDNA a quantitative PCR (qPCR) can be used by analyzing patients’ blood samples. In trials, using serum of patients with either colorectal [116] or lung cancer [117], it was shown that cfDNA levels were significantly lower after surgery and in case of colorectal caner, the cfDNA level were even more sensitive and specific than traditionally used tumormarker. cfDNA seems to be a very promising and highly applicable biomarker for diagnosis and monitoring of specific malignant disease. Apoptotic EVs, those extracellular vesicles created by disassembly of apoptotic cells, can be detected by using flow cytometry or the electron microscopy [58]. Although EVs from healthy cells are distinctly smaller than the large apoptotic bodies, there still exists a size-overlap with aEVs. This circumstance impedes a clear allocation of ApoEvs to apoptosis. Circulating free HMGB1 is usually quantitated by an ELISA. It measures total free HMGB1 and cannot differentiate between the three oxidation forms of HMGB1. They induce completely different immune responses. Free HMGB1 can derive from dying cells but also from activated macrophages. This has to be considered in the interpretation of the ELISA results. Extracellular microRNAs (ex-miRNA) are ribonucleoprotein particles which are stable over a long time in human plasma. Quantification of miRNA expression can be performed by using quantitative PCR. A potential disadvantage is a signal overlap with ex-miRNA released without relation to cell death, as exmiRNA can also be byproducts of regular cell activity. Proteins of the RIPK3-MLKL-pathway are proteins and can be detected easily with ELISA technology. Nevertheless, only the phosphorylated form of MLKL facilitates the membrane disassembling of a dying cell. Phosphorylated MLKL is usually quantitated by Western blotting. This is a time-consuming method. A recent paper of Dough Green group describes a pMLKL-specific ELISA wich will help to overcome this hurdle [118]. There are several studies that analyzed multiple circulating biomarkers of cell death in the same sample. For instance, Rack and colleagues compared the level of active caspase 3/7 with cell-free DNA, and circulating nucleosomes in breast cancer patients [30]. They found significant correlations of circulating nucleosome with DNA concentrations (p = 0.001) and nucleosome concentrations with page 22 of 48

caspase activities (p = 0.008). Of all cell death markers caspase 3/7 activity showed the best receiver operating characteristics (ROC) regarding the distinction between benign and malign breast cancer. Similarly, Fuchs and co-workers used caspase 3/7 activity, cell-free DNA, caspase‐cleaved cytokeratin‐18 (M30), and total soluble cytokeratin‐18 (M65) to detect cell death in colorectal cancer patients receiving chemotherapy [29]. All markers increased significantly on day 3 of the first treatment cycle. Cell-free DNA showed the highest increase. The group of Pamela Holland compared the time course of different circulating cell death biomarkers in non-small cell lung cancer (NSCLC) patients [119]. Serum samples were collected at baseline, 5, 24 and 504 hours after dulanermin treatment. Caspase 3/7 activity, ccCK18 and cell-free DNA showed similar time-dependent concentration curves. The levels increased within 5h and all were cleared at 24h. Furthermore, CRC and sarcoma showed a delayed time course in response to dulanermin treatment as compared to NSCLC. Taken together, these results show that different biomarkers for cell death correlate quite well with each other but their concentration depend on time and on storage time. The correct time point seems to be much more important than the method.

7. Conclusion and Outlook All circulating biomarker of cell death have a conceptual limitation regarding their sensitivity. Cell death, especially apoptosis, occurs constantly at high rate in a healthy body. The resulting dead cell remnants are cleared by phagocytes before any overflow of intracellular molecules into the circulation can occur. Thus, the continual process of physiological cell death is not detectable in the circulation. Disease-related additional apoptotic cells, which are cleared in time, are also undetectable. Only when the degree of cell death exceeds the clearance capacity, dying cells progress to secondary necrosis, i.e. to post-apoptotic disintegration of the cell membrane and the cell content is released. Consequently, all circulating biomarkers are only suitable to measure excessive cell death (or inhibition of clearance processes).

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It is barely feasible to draw any conclusions about the tissue of origin. For instance, an increase of circulating biomarkers in response to chemotherapy of cancer patients could derive from dying tumor cells or be the results of hepatotoxic side effects of the drug. Circulating ccCK18, however, can only derive from CK18 expressing cell types. Although many tissues express CK18, elevated levels of ccCK18 can be narrowed down to a definite organ under specific conditions. For example, CK18 is highly expressed in the liver. ccCK18 has been therefore proposed as specific biomarker for apoptosis of liver cells in studies of proven liver diseases such as hepatitis [43]. Of course, a concomitant cell death of other CK18 positive tissues cannot be excluded. Circulating nucleosomes with tumorspecific DNA or ex-miRNAs are other examples of cell death biomarkers which allow to draw conclusion regarding the tissue of origin. Also circulating aEVs can contain characteristic molecules that indicate their parent cells [59]. This field of research is progressing very fast and the technical possibilities to analyze extracellular vesicles are constantly improving. Therefore it is likely that this field of research will contribute to the identification of new and more specific circulating biomarker of cell death in the near future. Although some circulating cell death biomarker may give an indication of the tissue of origin, it is still mandatory to confirm this conclusion by histological analysis of the proposed tissue. Any contribution of other tissues to the overall biomarker in the circulation can never be ruled out with complete certainty. A very new and completely different, non-invasive method to detect disease related cell death is invivo-imaging using specific tracers [120]. An annexin-V labeled radionuclide is used as a novel tracer in combination with PET and single-photon emission computed tomography (SPECT). Annexin-V binds phosphatidylserine on the surface of dying cells. A major disadvantage of radiolabeld annexinV is its slow clearance and high background signal [121]. In spite of these limitations, 99mTc-annexin V was applied in a clinical trial with cancer patients receiving chemotherapy [122]. The 99mTcannexin V tumor uptake correlated with the outcome of therapy. The recently developed new tracer 18F-ML-10 is capable of selective targeting as well as binding, uptake, and accumulation within apoptotic but not in viable or necrotic cells [123]. Results of clinical studies in the near future will be awaited with interest. page 24 of 48

In conclusion, numerous studies confirmed that circulating cell death biomarkers can help to detect diseases in an early stage, to monitor response to therapy and to predict prognosis in patients. Their specificity for cell death is limited and a concurrent analysis of a complementary biomarker or a histological validation is strongly recommended. In addition, all studies showed that an accurate timing as well as an exact assay designing are crucial in the emerging field of cell death biomarker research.

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FIGURE 1 Biomarkers released from dying cells. Induction of apoptosis leads to an activation of caspase 3 (Casp3) which is a protease with hundreds of different substrates. Active Casp3 induces a rupture of the sub-membranous cortex leading to the formation of apoptotic blebs on the cell surface (1). They are finally released into the microenvironment as apoptotic extracellular vesicles (aEVs). aEVs are formed quickly after induction of apoptosis. When the cell is not cleared during this early phase the membrane becomes instable and disintegrates. This results in a leakage of intracellular components including activated Casp3 (2). Its proteolytic activity can be detected in the extracellular fluid. Intracellular active Casp3 cleaves the cytoskeletal protein cytokeratin 18 (CK18) into smaller fragments (3). One of them exposes a specific neo-epitope and can be detected as caspase cleaved CK18 (ccCK18) used in the M30 ELISA. The complementary M65 ELISA detects total released CK18 (i.e. cleaved and non-cleaved). During the early phase of apoptosis, the nuclear protein HMGB1 binds tightly to the chromatin. Active Casp3 enters the nucleus and degrades the chromatin into single nucleosomes. They are then released into the cell surroundings as nucleosomes and HMGB1 nucleosome complexes (4). Free HMGB1, however, indicates that this process did not take place and is therefore regarded as marker of accidental or regulated necrosis (5).

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TABLE 1 Circulating Biomarkers for Cell Death

cirulating biomarker

Type

enzymatic activity Caspases protein

Caspase cleaved CK18

protein

Nucleoso mes

DNAhistone complexes

cell-free DNA Apoptotic extracellular vesicles

HMGB1

exmiRNA

MLKL

DNA subcellular vesicles

protein

ribonucleoprotein particles protein

Method

Matrix

Limitation/Comment

enzymatic DEVDase activity test (fluorescence)

serum cerebrospinal liquor

Highly sensitive but no distinction between different caspases

ELISA

plasma serum

Unclear relation to cell death because of the missing discrimination between activated and total caspase

ELISA

plasma serum

Limited to CK18 expressing tissues, limited sample stability at -80°C

ELISA

PCR Flow cytometry Electron microscopy ELISA Immunohistochemi stry In situ Hybridization PCR ELISA PCR Immunohistochemi stry

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plasma serum

Affected by epigenetic modification of the DNA and post translational modification of histones

plasma serum blood other body fluids

Time consuming analysis, size overlap with EVs from healthy cells

plasma serum tissue

Free HMGB1 can also derive from activated macrophages

plasma serum serum tissue

Highly sensitive

Signal overlaps with exmiRNA released without any relation to cell death Analysis should focus on phosphorylated MLKL