Beyond basic research: the contribution of cathepsin B to cancer development, diagnosis and therapy

ABSTRACT

Introduction

In view of other candidate proteins from the cathepsin family of proteases holding great potential in being targeted during cancer therapy, the importance of Cathepsin B (CtsB) stands out as being truly exceptional. Based on its contribution to oncogenesis, its intimate connection with regulating apoptosis and modulating extracellular and intracellular functions through its secretion or compartmentalized subcellular localization, collectively highlight its complex molecular involvement with a myriad of normal and pathological regulatory processes. Despite its complex functional nature, CtsB is emerging as one of the few cathepsin proteases that has been extensively researched to yield tangible outcomes for cancer therapy.

Areas covered

In this article, we review the scientific literature that has justified or shaped the importance of CtsB expression in cancer progression, from the perspective of highlighting a paradigm that is rapidly changing from basic research toward a broader clinical and translational context.

Expert opinion

In doing so, we detail its maturation as a diagnostic marker through describing the development of CtsB-specific Activity-Based Probes, the rapid evolution of these toward a new generation of Prodrugs, and the evaluation of these in model systems for their therapeutic potential as anti-cancer agents in the clinic.

KEYWORDS:

• Cathepsins
• proteases
• apoptosis
• cell death
• cancer
• activity-based probes
• therapy

1. Introduction

The established regulation of the cathepsin (Cts) proteases in molecular cell biology is an area of research that has flourished over the recent 15 years. Whilst such proteases have always been recognized for their central role in lysosomal function, more recently, their effects outside of the cell, as architects of Extracellular Matrix (ECM) remodeling, have also become established. When coupled with some of their emerging intracellular functions, and the regulatory inter-relationships between these sub-cellular compartments, their collective biological effects and their amenability for therapeutic targeting are coming into focus in a number of disease pathologies, such as cancer [1,2].

The family of Cts proteases is composed of 15 members and which can be broadly sub-divided up into cysteine (B, C, F, H, K, L, O, S, V, Z/X, W), aspartic (D, E), or serine (A, G) proteases, from which some cysteine proteases have been reported to possess exopeptidase (B, H, Z/X, C) or endopeptidase (B, S, K, V, F, L) activities (reviewed in [3] and [4]). While normally resident in the lysosomes, the cathepsins can be secreted or leaked out from these under conditions of cathepsin overexpression, a drop in pH or upon alterations in the oxidation state of cells [5–8]. Moreover, they can also be expressed as protein-isoforms that can localize to the mitochondria or nucleus and regulate cellular proliferation and fate (reviewed in [9]). When taken with the enzymic activation of cysteine cathepsins being enhanced in a low pH-dependent manner, as with Cts-B, -K, and -L in vitro [8,10], these factors collectively highlight the importance of a number of key regulatory cues that dictate the spatio-temporal expression of cathepsins, and can originate from the metabolic programming of the host cells in which they reside [8].

When viewed from a broader perspective, the deduction that most of the cathepsins have been described as being good therapeutic target proteins, particularly in the field of cancer research, is therefore not surprising. This viewpoint has been largely shaped by them being overexpressed or secreted from a number of cancer cell types, macrophages, and fibroblasts, as seen (for example) during gastric cancer development (reviewed in [11]). Here, one other significant property being their enhanced proteolytic activation under relatively low pH conditions, which is synonymous with the hypoxic conditions that some tumors can thrive in [6]. In this context, cysteine Cts-B, -L, -D, and -S justify their candidacy as good therapeutic targets as some of these have been reportedly overexpressed and secreted [6] from a broad spectrum of cancers (reviewed in [3]) and exhibit enhanced intracellular protease activity against substrate proteins from the pro-apoptotic pathway such as BID [12,13] under relatively low pH conditions [8,14,15].

Consequently, some of the most promising anti-cathepsin targeting agents currently emerging have diversely taken the form of small-molecule inhibitors (SMIs) or peptide therapeutics, and both of which have demonstrated their effectivity and potential through their development as Activity-Based Probes (ABPs) for clinical diagnostic-testing purposes [16–18]. Importantly, such diagnostic probes have also topically evolved toward the generation of Photo-Sensitization-ABPs (PS-ABPs) and Prodrugs, either of which can be activated upon the cleavage of a CtsB-specific target sequence that liberates a diagnostic or therapeutic agent [19–21]. Such elaborate targeting strategies have been successfully conceived from structure-based findings, which are founded on the rationale that cathepsins are normally susceptible to inhibition, as seen between some cystatin inhibitor proteins and Cts family members, such as CtsB [22–24]. Consequently, through the diligent extension of such studies, a prolific area of research with a strong clinical trajectory has developed, which is coming to fruition through the development and testing of novel cathepsin protease-targeting therapeutic agents in model systems.

Herein, we focus on the relatively well-characterized CtsB protein, through highlighting its intracellular, extracellular, and functional properties that are central to cancer progression. We contextualize the clinical significance of such research findings and describe recent progress focused on how such findings have been adapted and harnessed for ABP-based diagnostic assay development and then extended for anti-cancer Prodrug therapeutic design.

1.1. Cathepsin B-isoform protein expression and cancer

Unlike most of the cathepsin proteases that have restricted tissue expression, Cts-C, -B, -H, -K, -O, -L, and -Z/X are expressed with relative broadness and which have remained at the forefront of research through unveiling and defining general mechanisms for cathepsin protein expression [25], activation, and substrate cleavage during normal cellular homeostasis or disease progression [26]. From such studies, CtsB has emerged with heightened importance due to its enhanced genomic expression and function in tumor metastasis [26,27] during advanced gastric or breast cancer (BC) [28,29], and in patients with enhanced inter-pulmonary metastases [30].

Under normal conditions, CtsB mRNA is ubiquitously expressed in a diverse range of tissues and highly expressed in liver, kidney, thyroid gland, and spleen as a protein of 339 aa from chromosome 8.p23.1 [25,31–33], and which can be up-regulated by IL-6 [34] during inflammation, and by transcription factor activation as seen with SP1 [35], USF [36] and possibly p53 [37,38], either directly or indirectly upon p53-responsive miRNA expression (reviewed in [39,40]). More recently, CtsB expression was reportedly enhanced by transcription factor MZF1 in BC cells [41] and the secretion of which is also enhanced during melanoma invasiveness in an Ets1-, SP1-, NF-kB- transcription factor-dependent manner [42]. Consequently, its overexpression in disease is believed to contribute to the progression of hepatocarcinoma [43], colorectal cancer [44], esophageal cancer [45], BC [38,46], lung cancer [47], squamous cell carcinoma [48], liver metastases [49], gastric cancer [50], glioblastoma [51], and even osteoarthritis [52], as confirmed by a number of CtsB-directed expression, antisense [53], siRNA, and shRNA studies [54]. However, robust CtsB expression may not strictly correlate with the classical stage-specific manner of cancer development, and may be a feature related to cancer type. This is largely based on a number of recent studies (reviewed in [55]), where a significant number of patient colonic tumors expressed CtsB irrespective of their stage of development, whereas the remainder tested negative for CtsB expression [56]. Conversely, another study addressing CtsB expression-trends in colorectal cancer reported that the expression of this protease contributed to colorectal cancer development and invasiveness, and reported enhanced expression of CtsB much earlier during cancer development, which declined during the later stages [57,58].

With these observations in mind, it is worth highlighting that most of these studies have been aimed at reporting the expression levels of full-length CtsB [59]. However, based on a number of in-silico and experimental sources (reviewed in [9]), full-length CtsB can also be co-expressed as a number of protein isoforms [60–63], which lack the secretory signal (CB-2) or the secretory signal and part of the cathepsin pro-domain (CB2-3), as a result of the alternative splicing of the CtsB transcript. Importantly, expression profiling studies have linked CB-2 [61,64] (reviewed in [9]) and CB2-3 overexpression to kidney, liver, colorectal cancers and alternatively to the progression of osteoarthritis [65], thus highlighting their potential involvement in a diverse number of diseases and their growing exploitation as diagnostic markers. Furthering such elegant studies to also incorporate other cathepsin protease isoform members does offer greater potential in defining novel biomarkers, whilst also unveiling their mechanistic involvement in established disease-specific regulatory paradigms. This may be of great significance, particularly as Cts-C, -E, -L, -M, -S, and -W are also expressed as potential isoform proteins (reviewed in [9]).

In summary, enhanced CtsB isoform expression is linked to the development, and progression of a number of cancer types and the key signaling pathways or transcription factors responsible for this are indeed being identified and functionally dissected. However, the intermolecular-regulatory relationships at the compartmentalization level (Figure 1), and the precise biological function of each CtsB isoform protein, during cancer development or progression does remain to be explored more fully [66].

Figure 1. The protein expression, trafficking, and targeted detection of cathepsin B (CtsB). Transcribed CtsB is translated and enters the secretory pathway and upon glycosylation resides in perinuclear lysosomes. Under specific cell stimulatory conditions or upon overexpression, CtsB can translocate to peripheral lysosomes and is secreted, where it can modulate extracellular matrix dynamics. Alternatively, CtsB can leak from lysosomes, process apoptotic proteins such as Bid and BAX which contribute to mitochondrial outer membrane permeabilization (MOMP) and apoptosis. CtsB can also reside in the nucleus of some cells as a result of it leaking from lysosomes as a mature protease, or after being expressed as a non-secreted CtsB isoform protein. Quench fluorescence Activity-Based probes (qABP), Photo-Sensitized ABPs (PS-ABP), or Prodrugs have been designed and tested to detect or target compartmentalized mature CtsB within the Extracellular Matrix (ECM) or within the intracellular endo-lysosomal compartments.

1.2. Cathepsin B subcellular localization and function

As touched upon, the expression of cathepsin proteases can alter the fate of cells and how they might respond to natural extracellular signaling cues, under normal conditions or during disease progression. Based on this line of thought, a number of interesting avenues of research have developed over the recent years, which have allowed a fuller picture to develop with regard to how cathepsins can modulate cancer progression based on their subcellular and extracellular localization (reviewed in [3,9]). Here, the main avenues of research have revolved around defining how the lysosomes lose cathepsin proteases to the cytoplasm (or nucleus) and how cathepsin protease secretion can be enhanced from the lysosome into the extracellular milieu (Figure 1).

Under normal conditions, the cathepsins come to reside in the perinuclear lysosomes following their passage through the late endosomes, where a drop in pH permits their intermolecular activation and pro-domain removal as with CtsB [67], and which can also be mediated by CtsG [68]. However, under certain disease pathologies, as in malignant BC progression [69], CtsB comes to reside in peripheral lysosomes and in exocytic vesicles [5] as a location believed to contribute to enhanced pro- and active-CtsB secretion. Here, one physiological trigger for this includes extracellular acidification and which can be simulated by exposing cells to conditioned media, as seen for BC cells [5,6]. Mechanistically, the expression of USP17 can also enhance Cts secretion, as reported for lysosomal trafficking of CtsD to the cell periphery in response to EGF signals, and offers clues about whether this process also modules CtsB protein trafficking [70].

To complement such findings, Lysosomal Membrane Permeabilization (LMP) or Lysosomal Leakage (LL) is additional mechanisms by which the cathepsin proteases can exit lysosomes and enter the cytoplasm, where they can exhibit unique biological effects based on their nuclear and mitochondrial localization, for example. As an alternative mechanism, this may arise through the generation of a novel mitochondrial localization signal as a result of alternative transcriptional splicing events, and as reported for CtsB [71] (reviewed in [9]). In the context of cancer cells, the lysosomes are reported to be abnormal, larger and more fragile than normal, thus elevating their sensitivity to agents and mechanisms that can induce LMP through cathepsin overexpression and LAMP1/2 cleavage [2,72], reactive oxygen species [73], metal ions [74], inflammatory cytokine signals [75], inhibition of sphingokinase-1 or S1P loss [76,77] and by lysosomotropic agents [78], such as Siramesine [79,80], Riccardin D (RD-N [81]), Nortriptyline [82], or carbon black nanoparticles [83]. Importantly, such agents have the potential for enhancing cathepsin-mediated cell death, as reported for CtsB nuclear relocalization, after RD-N mediated LMP induction, and by elevating DNA damage and suppressing BRCA1 activity [81,84]. Similarly, LL-derived CtsB has also been associated with nuclear sirtuin 1 degradation during the aging process of the mouse brain, giving rise to impairments in cognitive function [85]. Alternatively, the loss in expression of CtsB can reduce proliferation and elevated IL-17A/SAA-induced HaCat keratinocyte differentiation [86]. Such reports collectively support a central role for CtsB expression in cellular growth and differentiation in response to extracellular signals.

More recently, a growing biological feature of Cts proteases is their localization in the nucleus, as reported for Cts-D [87], -B [81], -H [88], -L [89–91], and more recently -S [92] (reviewed in [9,12]). Among these nuclear cathepsins, CtsB has been reported to cleave BRCA1, in prostate cancer cells with the effect of sensitizing cells to the effects of Cisplatin [81]. Through alternative transcriptional splicing, cytoplasmic CtsB expressed in the absence of its amino-terminal secretory signal (or CtsB derived from cellular LMP) has been reported to reside in the nucleus in proliferating thyroid cancer cells, where it can cleave histone protein H1 [93]. Unlike other CtsB isoform proteins, which can also localize to the mitochondria [94] and induce mitochondrial dysfunction [95], these nuclear-resident CtsB molecules arrive at the nucleus in the absence of them encoding a bona fide nuclear-localization signal [93]. Conceivably, leaked CtsB can be chaperoned into the nucleus, and the mechanism for which needs further elucidation, based on it potentially being harnessed for therapeutic targeting purposes, especially during chemoresistance (Figure 1). Such a perspective is largely shaped by studies demonstrating the nuclear relocalization of Cts-L and -D, by their respective chaperones, the CUX1/Snail protein complex [90,96] and the protein BAT3 [87], which can induce the proliferation or trans-differentiation of cells.

In summary, a number of potential underlying mechanisms and effects have been reported for the existence of nuclear cathepsin proteases, which could offer additional research avenues that can be exploited for compartment-specific therapeutic targeting and diagnostic strategies.

1.3. Cathepsin B regulation and substrate specificity

Broadly speaking, CtsB-specific substrates can be classified into two groups based on the localization of the protease. Whilst considerable focus has been directed at the effects of protease-directed extracellular remodeling during tumoral expansion, intracellular cathepsin expression can also contribute to this by cleaving a number of cancer-related substrate proteins, which modulate cellular proliferation and resistance to apoptosis (reviewed in [12]). This may have particular relevance during the acidification of cells or their microenvironment, the latter of which may arise due to metabolic reprogramming of cancer cells [97,98], and which can collectively give rise to enhanced cathepsin expression, secretion [7], and subcellular relocalization [6].

Extracellularly, CtsB has been seen to act on modulating the architecture of the ECM through the cleavage of E-cadherin (as reported for human pancreatic cells [99]) and other ECM components, such as Fibronectin, Collagen IV [100], and Tenascin [101], in bovine lens capsules, astrocytomas, or glioblastoma cells, and during tumor development, invasiveness, and progression. Here, the regulation of angiogenesis or tumor vascularization by cathepsin proteases has also gained considerable attention through their potential to be targeted as part of emerging therapeutic strategies. In this context, Cts-S and -L-mediated cleavage of COLXVIII to yield the anti-angiogenic factor endostatin has taken on a degree of importance during cancer progression and development [102]. Additionally, CtsB expression can promote tumor angiogenesis [103] through CD18 shedding from leukocytes [104,105] or negatively regulate angiogenesis, through inhibiting VEGF-A expression [106]. As an area that requires further exploration, such reports collectively support the ever-growing connection between the cathepsins and cytokine (or receptor protein) shedding, and how such events modulate the ECM to favor tumoral expansion and its metastatic spread [107].

Intracellularly, the importance of lysosomal factors in regulating cell fate emerged from the observations that lysosomal rupture induced cell death, hence this organelle being colloquially named the ‘suicide bag’ [108,109]. At the molecular level, Cts-B and -D were seen to cleave and activate BID [110], following the leakage of these proteases form the lysosome prior to caspase-8 activation [13,111–113] Figure 1. As this is a mechanism that requires the partial cleavage of BID to active ‘t-BID,’ it is likely to occur through a stringently regulated mechanism, which permits BID cleavage in the absence of complete BID degradation [13,114,115]. One such mechanism could be the pH-dependent activation of CtsB. Here, at a normal intracellular pH, the cysteine cathepsins are relatively less active, which could account for why the partial cleavage of BID is permitted, and give rise to cellular apoptosis through the intrinsic pathway upon LMP induction [13]. In support of these findings, BID cleavage has been mapped to Arg-65 or Arg-71, through the activity of Cts-B, -H, -L, -S, and -K, which occurs close to the preferred cleavage site of caspase-8 [110]. Conversely, under lower pH conditions, LMP-derived or secreted cathepsins can exhibit enhanced activity, giving rise to the complete degradation of apoptosis-regulatory proteins, and certain extracellular ECM components [6]. Here, Droga-Mazovec et al. (2008) reported the positive cleavage of BAK (along with Bcl-2, Bcl-xL, and Mcl-1 proteins) at pH 7.2 by Cts-B, -L, -S, and -K in vitro [116] and Soond et al. (2021) reported the cleavage of the BAX protein, by CtsS at pH 7 and 5 [92]. Whilst the cleavage of such protein substrates may alter cellular fate, the effects of CtsB and its lysosomal cleavage of the cell cycle inhibitor p27^KIP are also worth mentioning as a contributing factor to tumor progression, in light of elevated cyclin B1 levels being reported in CtsB-deficient tumors, thus linking positive CtsB expression with cellular proliferation [55].

Collectively, such findings highlight a pH-dependent mechanism for CtsB release, activation, and the cleavage of substrates that are central to determining cell fate and growth. Moreover, such fluctuations in pH may also have the likelihood of altering CtsB-directed substrate and therapeutic specificity [117].

1.4. Cystatin-mediated inhibitory regulation of cathepsin B

Clearly, the emerging pathological significance of cathepsin proteases over the years has been established on their overexpression in certain disease states, leakage from the lysosomal compartment, and their subsequent ability to cleave key protein intermediates central to determining cell fate [3]. Based on the evidence derived from subcellular compartmentalization of cathepsin proteases and substrate specificity, another key regulatory step connected with regulating the cathepsins originates from the actions of the reversible cystatin inhibitor proteins [22,118,119].

As a superfamily of proteins, the cystatins can be subclassed into clans, families, and Type 1–3 subfamilies. Here, the Type 1-cystatins (∼100 aa, or stefins) are produced intracellularly [120] and can also be secreted [121]. Type 2-cystatins (∼115 aa) are exclusively secreted [122], whereas the Type 3-cystatins are secreted into the blood and are of varying molecular weights and are also referred to as Kininogens [118]. The combined data from a number of excellent structural studies has yielded valuable information about how cystatins modulate the cathepsins, whilst others have described how the cathepsin proteases are regulated by their inhibitory pro-domains [23]. When combined, both schools of thought have unveiled a number of robust regulatory mechanisms, which highlighted the susceptibility of cathepsins to therapeutic targeting [26].

Generally speaking, the cystatin inhibitors, which are reversibly bound to the active cathepsin proteins, can be displaced upon the recognition of substrates by the cathepsin protease [26], as seen with stefin A displacement from CtsB [24]. Here, CtsB is viewed as being unique in the sense that it encodes a ∼20 aa occluding loop motif [123–128], from which residues H110 and R116 bind the main body of the enzyme through residues D22 and D224, an interaction that restricts access to the active site cleft and offers CtsB carboxypeptidase activity through residues H110 and H111 residues docking with the carboxyl group of the substrate. Mutation analysis of these key residues have been reported to enhance CtsB endopeptidase activity [129]. The flexibility of the occlusion loop also offers regulatory function as deletion of aa 109–118 can abrogate CtsB exopeptidase activity, and yields a protease harboring endopeptidase activity alone [130]. Collectively, these observations support a mechanism that involves occluding loop displacement from the active site cleft by the incoming substrate, thus allowing CtsB to act as an endopeptidase. Similarly, an alternative CtsB inhibitory step has also been suggested, which involves the insertion of the CtsB pro-domain into the active site cleft upon maintaining the inactive zymogenic form of CtsB [23,131,132]. Importantly, this has been suggested to occur in competition with the occlusion loop, which competes more effectively at a lower pH [133]. Collectively, full activation of CtsB through unveiling its active site has been proposed to occur upon the cleavage of the intermolecular pro-domain by other cathepsins during secretion [131], upon a drop in intracellular pH [133], exposure to glycosaminoglycans, or upon reaching the cell surface under decreased pH conditions and in a V-ATPase-dependent manner [5,7,68,134,135].

1.5. Cathepsin B is a cancer-specific diagnostic marker

Based on observations that CtsB overexpression can be correlated with cancer progression, such reports have paved the way for accurately quantifying CtsB levels for tumor identification, and cancer diagnostic or prognostic assay development purposes. Here, CtsB has been reported to be overexpressed in a diverse number of cancer types of the pancreas [136], lung [30], breast [137], colon [138], glioblastoma [51], hepatocarcinoma [66,139], squamous cell carcinoma [48], gliomas [140], head and neck cutaneous squamous cell carcinoma (mHNcSCC [141]), pediatric AML [142], and malignant melanoma [143]. Whilst such observations do highlight the potential for CtsB to be promisingly earmarked as a potential diagnostic marker, the robustness and accuracy of this test has seen developments through the co-detection of CtsB alongside other biomarkers of interest. For example, CtsB and CtsD transcripts and proteins were simultaneously detected in squamous cell carcinoma cells [48], and liver metastases from colon adenocarcinomas [49].

In a similar context, the co-detection of CtsB alongside cystatin proteins has also been exploited for their diagnostic potential and helped to validate cathepsin expression levels as reliable diagnostic markers. For example, CtsB and cystatin C ratios were quantified from colorectal cancer patients [44] or sera from esophageal cancer patients [144], and ratio levels found to be significantly higher in comparison to normal. Similar findings in support of CtsB levels being significantly higher than stefins A and B in hepatocellular carcinoma (HCC) tissues were also reported by Lin et al. (2016), supporting a correlation of CtsB expression and HCC progression whilst establishing the use of CtsB and stefins A and B as good diagnostic markers [43]. In a more recent study, elevated CtsB and stefin A levels were reported for renal clear cell carcinoma tissue samples, thus validating the cancer-specific nature of such correlation-based testing regimens [145].

Collectively, these diagnostic approaches do offer significant degrees of accuracy in adopting the use of CtsB (±cystatins or stefins) as diagnostic assays in a variety of cancer types. Whilst limitations in accuracy may arise from the abilities of cathepsins to degrade cystatin proteins, and for cathepsins (or cystatins) to be sub-compartmentalized in the cytoplasm and the nucleus (to varying degrees), an alternative repertoire of emerging Activity-Based Probe (ABP)-tests has been developed. Such rapidly emerging approaches are permitting the limitations arising from adopting the above classical approaches to be addressed, with greater speed and accuracy.

2. Cathepsin B can be detected and therapeutically targeted with ABP, qABP, PS-ABP, and fluorescent prodrug technology

Derived from co-crystallization structural studies of CtsB and stefin A [24] and through targeting CtsB catalytic activity by the screening of peptide libraries and peptide cleavage studies, a number of useful diagnostic and therapeutic peptides or small-molecule inhibitors (SMIs) have been arrived at [146,147] Figure 1. Importantly, due to the highly conserved structural nature of the cathepsin active sites, such approaches have successfully yielded or validated candidate therapeutics that can specifically distinguish between Cts-B, -D, -H, and -L, using site directed mutagenesis of the protease active- or substrate cleavage-sites [148,149].

Consequently, in a shift from the classical ELISA, RT-PCR, or IHC paradigms for detecting CtsB protease expression, emerging assays have incorporated the use of irreversible fluorescently labeled Activity-Based Probes (ABP) or substrates, with some very encouraging outcomes. Simply described, these are composed of a fluorescently labeled ‘warhead’ conjugate molecule, which can form a covalent bond with the active-site thiol group of the cysteine protease [150], thus allowing its deactivation and detection (Figure 2). As a good starting point, small-molecule probes linked to the warhead and fluorescent conjugates specific to cathepsins did have the outcome of validating a ‘proof of principle’ in being able to detect proteases using optical fluorescent imaging techniques such as Fluorescence Tomography (FT), Positron Emissions Tomography (PET), or Near InfraRed Fluorescence (NIRF). Such approaches have been developed and superseded by ABP substrates (Figure 2), which offer greater sensitivity and specificity, seeing as the human genome encodes over 500 proteases [151]. Such substrates were demonstrated to target CtsB by the protease recognizing a CtsB active site-specific peptide sequence, and which have also been referred to as ‘smart probes,’ particularly in the context of qABP (quench fluorescence-ABP) substrates (Figure 2). However, in the absence of an active-group thiol-targeting warhead, such substrates do not inhibit CtsB and leave the targeted protease active [16]. Nevertheless, such approaches have yielded successes, through the development of PS-qABP (photosensitized-qABP), which rely on the interplay between their three non-toxic components comprising two fluorophores linked through a cathepsin cleavage site. Following the cleavage of the agent, illumination at a particular wavelength of light can give rise to the generation of cytotoxic reactive oxygen species (ROS) from endogenous oxygen. Similarly, the advantages of targeting the cathepsins in such a manner also permit the delivery of Prodrugs to the site of the tumor and the cleavage products of which can also be fluorescently detected using FT/NIRF [21]. Generally, PS-qABPs and Prodrugs can be generated through self-assembly or can be coupled to a carrier, which can enhance uptake of the Prodrug or release of the active drug (Figure 2). Such a technology has been evaluated for efficacy in cell line and xenograft models, some of which are showing some very encouraging outcomes.

Figure 2. Utilization of Cathepsin B (CtsB) detection or activity in cancer diagnostic assays or therapy. Activated-Based Probes (ABP), designed with a warhead moiety (read arrowhead), a CtsB recognition/cleavage sequence (such as GFLG, white box), and a fluorescent excitable probe (green circle) allow CtsB detection. The fluorescent tag can also be quenched by an accompanying chemical moiety, as in qABP substrates (pale green circle). PS-qABPs are composed of an excitation-detectable fluorescent tag (green circle) and a specific chemical intermediate (yellow circle), which inflicts cytotoxic effects can be released upon photosensitization. Fluorescent Prodrugs contain a CtsB cleavage site, which determines the release of an active drug (yellow circle) and a fluorescent tag for detection purposes (green circle).

2.1. Detecting CtsB expression with ABPs

As described in Table 1 for ABP Probes, visualization of GB123 in mice bearing human MDA MB-231 breast cancer tumors, followed by standardization of fluorescence against tumor weight [152], saw the accumulation of GB123 at 12 hr post-injection (PI). In comparison to a commercially available substrate (ProSense 750), GB123 was shown to exhibit 10–12× detected signal strength at 24 hr (PI). At the tested dose (and as a potential drawback), GB123 was observed to only label a subpopulation of cathepsins, leaving most active enzymes intact and uninhibited. In C2C12-derived mouse tumors, which express higher cathepsin protease activities [16], similar signals were obtained for GB138 in comparison to the commercial control, ProSense 680. In support of these encouraging findings, the specificity of GB138 was positively confirmed using gel-labeling experiments to detect Cts protease activity from GB138 treated tumor-derived whole-cell lysates. Analysis of tumors ex vivo showed a high uptake, whereas a low specific uptake was recorded in all organs except the lungs.

Table 1. Activity-Based Probes (ABPs) specific for detecting cathepsin proteases are highlighted. ABPs were tested for specific cathepsin protease detection using Gel-Labeling assays, cellular imaging approaches or in whole animal systems. The diseases that the ABPs have been tested for are also highlighted. NA-Not Applicable; Y-Yes; Mac-Macrophages; OA-Osteoarthritis; BC-Breast Cancer; K-Kidney; M-Mouse; AS-Atherosclerosis; KidC-Kidney Cancer.

ABP probe	Target Cathepsin	Warhead	Gel Labelling	Cell Imaging	In vivo Testing	Disease	Article
GB123	All Proteases and B, L, S	Acyloxymethyl ketone	NA	NA	M	BC	[16,152]
GB138	All Proteases and B, L	Acyloxymethyl ketone	Y	NA	M	BC	[152]
GB123	All Proteases and B, L, S	Acyloxymethyl ketone	NA	Mac	M	AS	[16,153]
GB123	B, S	Acyloxymethyl ketone	Y	OA	NA	OA	[154]
KDA-1	B	Fumarate diamine	Y	BC, K	NA	BC, KidC	[155]

The detection-potential of such agents has also been extended to other disease models such as atherosclerosis, where high levels of Cts-B and -S have been reported for macrophages derived from carotid plaques [153]. Using Fluorescent Molecular Tomography, GB123 could detect atherosclerotic (AS) plaques in mice and was confirmed by ex vivo analysis of tissues and macrophage labeling experiments. Similarly, Cts-B and -S were detected in chondrocyte cells and human Osteoarthritis (OA) tissue samples, and late OA sera samples [154]. Finally, taking a more cathepsin-specific approach, Bhuiyan et al. (2021) developed KD-1 (and cell-permeable derivative, KD-1-OMe) as CtsB-specific ABP probes, which inhibited the proliferation of highly metastatic MDA-MB-231 breast cancer cells and also detected CtsB in HEK293 cells [155].

Collectively, such findings describe the development and partial evolution of ABP probes through the GB123 paradigm, and the application for CtsB detection in numerous disease and cell types, whilst emphasizing the power of such probes based on their cellular (and tissue) uptake, relative signal intensity, and cathepsin specificity. Moreover, such a technology has the demonstrated potential for detecting CtsB from serum samples in a rapid testing assay, whilst simultaneously demonstrating the inhibitory effects of some of these probes, as in the instance of KDA-1 [155].

2.2. Detecting Cts B expression with qABP substrates

Moving forward, the approaches defined by the development of ABP probes yielded the generation of qABP probes and substrates [19]. For the latter, detection of specific cathepsins based on their ability to cleave a specific peptide connecting two parts composed of a fluorescent tag and its quenching moiety, was permitted.

For example, Bender et al. (2015) developed BMV083 and BMV157, the latter of which gave optimized CtsS specificity upon altering the quencher group to sulfo-QSY21 [156]. BMV157 (like EM503) could be used to visualize cathepsins in living cells and organisms, with the notable difference that EM503 had a broader range and better quenching abilities based on it using BODIPY-FL and the quencher BHQ-10. Whilst EM053 was not tested in mouse models for its effects on bone marrow-derived (BMD) cells and cancer proliferation, it could efficiently detect CtsS in living cells [156]. In support of such findings, through adopting similar experimental approaches, GB137 was demonstrated to successfully detect Cts-S and -B from macrophages and carotid plaques in atherosclerosis mouse models, whilst also demonstrating general applicability of such an assay for diagnostic purposes in diseases other than cancer [153]. Another promising example of a reagent with negligible toxicity was reported by Sun et al. (2019) for reagent JF1, which could be taken up by CtsB-expressing MCF7 cells. Importantly, JF1 could be used to visualize BC tumors, 10 minutes PI, demonstrating the rapid screening potential of this assay. Importantly, histological analysis of major organs showed zero toxicity following 1 week of treatment [157]. Finally, Probe 4 [158] and AS1411-Ce6-CQDs [159] could penetrate prostate, breast, cervical, and kidney cancer cells and successfully detect intracellular CtsB.

2.3. Harnessing CtsB expression for targeting cancer cells using PS-qABPs

As a significant and progressive development, PS-qABP and PDT have been utilized to target cell types over-expressing specific cathepsins using real-time imaging in living cells and in whole animals [20].

As highlighted in Table 3, CtsB-cleaved PMB1/PMB2 releases phthalocyanine units, which become cytotoxic upon their irradiation with light at a wavelength of greater than 610 nm and the effects of which were sensitive to the cathepsin inhibitor CA-074Me [160]. Prior to cathepsin-mediated cleavage, these PS-ABPs showed little background levels of fluorescence, and were readily taken up by cells [160]. Alternatively, with CTSB-PPP, no dark toxicity or ex vivo vascular function was observed with concentrations of 6 µM and was taken up by live BMC cells quite readily after 60 minutes [20]. Dose-dependent cytotoxicity and concomitant increases in the production of reactive-oxygen species (ROS) were observed with treated BMCs and upon their illumination with light sources of 671 nm. Similar trends and observations were also reported for Pep-SQ@USPIO using TNBC cells in mice bearing BC tumors using MRI/NIRF imaging. Here, following laser irradiation of Pep-SQ@USPIO, significant Tumor Inhibition Rates (TIRs), low Ki-67 expression, and high apoptosis rates were reported [161]. Finally, NPs@Ce6 approached the use of such technology through innovatively linking photosensitizers to a CtsB-Paclitaxel Prodrug. This approach utilized a two-stage process that involved photochemical internalization and then chemo-photodynamic therapy. Here, therapeutic uptake was rapid, showed greater than 98% inhibition of human bladder cancer growth, and using bioinformatic approaches the authors demonstrated cell cycle inhibition, increased ROS production, and reduced cell metastasis and invasiveness [162].

Table 2. Quenched Activity-Based Probes (qABPs) specific for detecting cathepsin proteases are highlighted. qABPs were tested for specific cathepsin protease detection using Gel-Labeling assays, cellular imaging approaches or in whole animal systems. The diseases that the qABPs have been tested for are also highlighted. NA-Not Applicable; Y-Yes; BMM-Bone Marrow Macrophages; Mac-Macrophages; M-Mouse; BC-Breast Cancer; AS-Atherosclerosis; PrC-Prostate Cancer; CerC-Cervical Cancer; KidC-Kidney Cancer.

qABP	Target Cathepsin	Warhead	Gel Labelling	Cell Imaging	In vivo Testing	Disease	Article
BMV083	B, L, S	acyloxymethyl ketone	Y	RAW264.7	NA	NA	[156]
BMV157	S	acyloxymethyl ketone	Y	BMM	M-4T1 tumors	BC	[156]
EM053	B, L, S, X	phenoxymethyl ketone	Y	BMM	NA	NA	[156]
GB137	B, S	acyloxymethyl ketone	NA	Mac	M	AS	[153]
JF1	B	-	NA	MCF7	M	BC	[157]
Probe 4	B	Cbz-Lys-Lys(2,4-DNP)-PABA	Y	DU145, MDA-MB-231	NA	PrC, BC	[158]
AS1411-Ce6-CQDs	B	GRRGKGGC cleavage	NA	HEK293, HeLa	NA	CerC, KidC	[159]

Table 3. Photosensitization quenched Activity-Based Probes (PS-qABPs) carrying specific cleavage sequences for Cathepsin B (CtsB) are highlighted. PS-qABPs were tested for specific CtsB detection in cells in vitro, and in vivo using whole animal systems. IC₅₀ concentrations are highlighted as are their abilities to inhibit tumor proliferation. The diseases that the qABPs have been tested for are also highlighted. NA-Not-Applicable; M-Mouse; H-Human; Lun-Lung; Liv-Liver; PTX-Paclitaxel; TNBC-Triple-Negative Breast Cancer; BladC-Bladder Cancer; Xeno-Xenografts.

PS-qABP Substrates	Cathepsin-Specific Sequence	In vitro	In vivo	Cancer	IC50	Tumor Inhibition	Article
PMB1/2	B (GFLG)	A549,HepG2	NA	Lun, Liv	0.21–0.39 μM	NA	[160]
CTSB-PPP	B (GAGRRAAG)	Bone Marrow Cells	NA	NA	12 µM (71.7 ± 7.9% reduced viability)	NA	[20]
Pep-SQ@USPIO	B (GFLG)	MDA-MB-231, MCF7	M	TNBC	100–800 µg/ml	83.5 ± 8.7% (Tumor Inhibition)	[161]
NPs@Ce6	B (GFLG)	T24, Spheroids	H-M Xeno.	BladC	0.16 μg/mL (PTX/Ce-6)	5x Reduction Tumor Volume	[162]

Collectively, such assays highlight the viability of PS-qABP as a substrate for the detection and treatment of tumors, and which may also show great potential in treating cancer-derived metastases or chemo-resistant cells.

2.4. Harnessing CtsB expression for targeting cancer cells with Prodrugs

To date, excellent studies (and articles) have been published showing the development of anti-cathepsin therapeutics, as in the case of ‘DARPin’ technology-against non-active site directed CtsB detection in mouse BC models in vivo [163], DOFA-which acts by targeting the occluding loop, thus obstructing substrate-active site access [164], CA-074Me-which acts by blocking the substrate-binding site of CtsB and reported as far back as 1991 [165], SMIs designed against CtsB using structure-based docking studies [166] and the incorporation of cathepsin-derived cleavable target sequences within Prodrugs for CtsB-targeted release, and areas that have all developed over the last 3 years.

As seen from Table 4, there has been consistent progress in the latter of these areas, through developing drugs such as pro-Doxorubicin or Eribulin in forms that self-assemble or can be loaded onto nano-carriers. Here, nano-carriers were viewed to help improve localized Prodrug delivery, release, and inhibitory rates in cell lines or mouse xenograft models. As reported, Doxorubicin was reported to have shown the greatest IC₅₀ effect at 9.11 µM and 0.86 µM against ovarian cancer cells [167] and fibrosarcoma cells [168], respectively. In xenograft mouse models, 90% tumor inhibition was reported for lung and liver cancers [169] and up to 83.1% inhibition of colorectal cancers [170]. From these findings, it is interesting to note the effects attributed to the choice of delivery nano-carrier, particularly in relation to the utilization of gold nanorods that gave up to 80% inhibition against lung and breast cancer development [171]. In this context, these differences could have arisen due to nano-particle toxicity, non-specific or rapid (and inconsistent) release of therapeutics, and possible mass-production/scale-up inconsistencies during the nano-carrier preparations.

Table 4. Selected Prodrugs are highlighted along with specific cleavage sequences for Cathepsin B (CtsB). PS-qABPs drugs were tested for their inhibitory effects in vitro cellular systems and in whole-animal systems. DOX-Doxorubicin; MIS-Minimal Inhibitory Sequence; M-Mouse; NA-Not-Applicable; BC-Breast Cancer; FibS-Fibrosarcoma; TNBC-Triple-Negative Breast Cancer; ColC-Colon cancer; Card-Cardiomyocytes; PanC-Pancreatic Cancer; PrC-Prostate Cancer; LuC-Lung Cancer; Pan-Pancreatic Epithelial; Ova-Ovarian Cancer; CerC-Cervical Cancer; GasC-Gastrointestinal Cancer; Endo-Endothelial; LivC-Liver Cancer; NSCLC-Non-Small Cell Lung Cancer.

Prodrug	Cathepsin-Specific Sequence	Drug/Nanocarrier	In vitro	In vivo	Cancer	Inhibitory Effect	Article
FRRG-DOX	B (FRRG)	DOX/ Self-assembling	HT29	M	ColC	62.4–83.1% reduction in tumor volume (1201 ± 297 to 315 ± 50 mm^3)	[170]
PNPs	B (FRRG)	DOX/Self-assembling	SKOV-3, HeyA8, MC38, CT26, H9C2, HDF, CCD-18Co	M	OvaC	IC50 9.11, 5.06, 8.98, 5.2, 111.36, 111.72, and 135.8 μM for SKOV-3, HeyA8, MC38, CT26, H9C2, HDF, and CCD-18Co cells	[167]
DPs-TPS4	B (FFKF)	DOX/ Self-assembling nanofibers	4T1, MDA-MB 231	NA	BC	NA	[172]
cPCPs	B (GAGRRAAG)	DOX/PEG	HT1080	NA	FibS	IC50 0.86 μM	[168]
GLFG/DOX	B (GLFG)	DOX/PEG Liposomes	HepG2	Z	LivC	GLFG/DOX inhibited cell viability by 60%	[173]
pHLIP-SS-GFLG-MSPLNPs@DOX	B (GFLG)	DOX/Mesoporous silica	A549, HepG2	M	LuC, LivC	90% (100 μg/ml)	[169]
Al-ProD	B (FRRG)	DOX/Albumin nano-vectors	MDA-MB231 H9C2	M	BC	7.33 µM and >200 µM for MDA-MB231 and H9C2 cells	[174]
AuNR Cluster	B (-)	DOX/Gold nano-rods	MCF-7, 16HBE	NA	LuC, BC	50%-80% (2.3 μg/ml)	[171]
MORAb-202	B (V-Cit)	Eribulin/ Maleimido-PEG2	Ovarian, Gastric and TNBC	M	OvaC, GasC, Endo, TNBC, NSCLC	IC50 20 pmol/L (for IGROV1)	[184]
BIMA,cath,KPA2	B (V-Cit)	BIM BH3 peptide/ PEG micelles	MEF, HeLa	M	CerC	~3-25 µM (50–0% viability)	[175]
PDMS-PMOXA	B (GFLG)	PAC/ Diblock-copolymer	OVCAR3	NA	OvaC	IC50 29.93 nM	[176]
Tf-MSN@AB	B (DEGFLGED)	Elevated Fe/ ROS/ Mesoporous silica	MCF7	M	BC	12–36% increased mortality	[177]
FA-GFLG-SN38	B (GFLG)	Folic acid/NA	SK-Hep-1, HeLa, Siha	NA	LuC	IC50 2–3 μM	[178]
r9-CatB-p14 MIS	B (GFLG)	P14 MIS/ NA	PK‐8, BxPC, HeLa, PC‐9, MCF‐7, T47D	M	PanC, BC, ColC, PrC, LuC	60–65% at 10‐μM	[179]
SMAC-FRRG-DOX	B (FRRG)	DOX-SMAC peptide/Self-assembling	MCF7	M	BC	Decreases in tumor volume to 71.5 ± 2.8%, 69.7 ± 3.33% and 58.6 ± 2.15% (14 d post-treatment)	[180]
PNPs	B (FRRG)	DOX (+Navitoclax)/Self- assembling	MDA-MB231	M	BC	PNPs+Navitoclax (76.97 ± 7.31% apoptotic) versus PNPs alone (47.76 ± 6.78%)	[183]
PD-NPs	B (FRRG)	DOX-anti-PD-L1 peptide/Self-assembling	HUVEC, H9C2	M	Card, BC	IC50 3.01 μM, 200 μM and >200 μM for 4T1, HUVEC and H9C2 cells	[187]
CAP-NPs	B (FRRG)	DOX (+anti-PD-L1)/Self-assembling	CT26, H9C2	M	ColC	50% complete regression compared	[186]
MTX–G–P4LDN–E2	B (GFLG)	Methotrexate-anti-EGFR peptide/P4LDN	MDA-MB-231	M	TNBC	82.6% reduced tumor volume	[181]
MORAb‐202	B (V-Cit)	Eribulin, Farletuzumab/ NA	OVCAR‐3‐A1, NCI‐H2110, A431‐A3, HL‐60, SJSA‐1	CM	BC	IC50 0.1 × 10^−10M, 7.4 × 10^−10M, and 230 × 10^−10M for IGROV‐1, NCI‐H2110, and A431‐A3 cells	[185]
HADD	B (GFLG)	Hyaluronic acid-Docetaxel/Self-assembling	MDA-MB-231	M	BC	High doses-99.71% tumor inhibition	[182]

Using a novel approach, some Prodrugs showed greater effectiveness when administered in a combined therapeutic regimen (in models systems), as self-assembling therapeutics, which can (for example) help address issues associated with low targeting efficiency. In this context, the efficacy of PNP-Dox administered with Navitoclax showed an increase in BC tumor inhibition to 76.97% in comparison to PNP-Dox alone (47.76% inhibition) [183]. Similarly, Eribulin/MORAb-202 [184] was effective at an IC₅₀ at 20 pM in a number of cancer cell lines and was seen to decrease further to 0.1 × 10⁻¹⁰ M, when co-administered with Farletuzumab against BC cells [185]. Other combined therapeutic regimens included the co-treatment of Doxorubicin with anti-PD-L1 -antibody [186] or -peptide [187] therapeutics, which enhanced IC₅₀ concentrations against colon and breast cancer development. Importantly, such approaches showed encouraging effects at concentrations with little in the way of side effects and non-specific toxicity or instability.

In summary, most of the studies report very encouraging outcomes based on cancer-specific efficacies, in a variety of in vitro or in vivo mouse model systems, the treatments for which can also be optimized and controlled based on the mode of therapeutic delivery.

3. Clinical and future directions for CtsB targeting

From scrutinizing the scientific milestones highlighted in Tables 1–4, what is apparent is that the area of PS-ABP (and Prodrug) development appears to be flourishing ahead of the research areas that incorporate the design and development of SMI anti-CtsB therapeutics. Whilst both strategies are not mutually exclusive in their mode of action, they can independently offer alternative and effective targeting approaches [188,189], essential for yielding robust Theranostics for the clinic.

Keeping with PS-ABP development, and upon scrutinizing the clinical trials database for keywords such as ‘cathepsin B’ (www.clinicaltrials.gov), we do find limited entries, but one of which does have great relevance (NCT03386942). Here, a CtsB cleavable linker conjugating Farletuzumab and Eribulin for the treatment of solid tumors [190] is utilized in the form of MORAb-202, which demonstrated promising anti-tumor activity in FRα-positive tumors at well-tolerated doses. Despite this encouraging output, the Maximum Tolerated Dose was not defined and so further studies are needed. From another completed study, LUMO15 (NCT01626066) was well tolerated for tumor detection in a cohort of BC patients, and the full outcomes of which are eagerly awaited. Encouragingly (from both studies) therapeutic and diagnostic agents tested for CtsB targeting are realistic scientific endeavors and which set strong precedents for such technological advancements to continue, particularly in the context of targeting other Cts proteases.

Underpinning such progressive and encouraging outcomes are the convincing basic research studies aimed at targeting cancer-specific CtsB expression, through adopting a number of in vitro and in vivo approaches, that utilize ABP-derived probes, substrates, and Prodrugs. Whilst some agents are readily taken up by cells and permit the detection of some tumor cells and their boundaries, other agents target cells or components of the ECM (such as CtsB) that collectively modulate direct tumor development from the stroma. Clearly, such hopeful and encouraging findings have surfaced through combining state-of-the-art in vitro and in vivo systems (Tables 1–4), and which are seen as sufficiently robust in permitting anti-CtsB agents to be characterized fully in a variety of cancer contexts, allowing the evaluation of important parameters, such as specificity, side effects, penetrability, half-lives, and tumor inhibition.

Clearly, with such rapid developments occurring, it may also be within the scope of such studies to target the biological effects of compartmentalized CtsB isoform proteins, as future studies evolve toward selectively targeting specific isoform proteins, at the subcellular or extracellular levels. Bearing in mind that some cathepsins and their isoforms do have clear input to chemoresistance, the above targeting approaches do consequently take on broader significance in offering better targeting specificity.

4. Conclusions

Over the last 3 years, the cathepsin proteases and their clinical applications have made great gains in the field of cancer research. Whilst they were originally thought of as proteases that had an important function in cellular homeostasis, their biological effects have been furthered as important players in a number of key regulatory mechanisms that span cell proliferation, survival, differentiation, and ECM regulation. As a versatile member of this family of proteases, clearly cathepsin B has been seen to have gathered heightened importance based on the wealth of knowledge that has been gained from its utilization as a diagnostic and prognostic marker for a number of cancers, and from it being therapeutically targeted and tested in preliminary clinical trials. When taken together, CtsB is clearly a protein that is shaping the paradigms through which we view this family of proteases from a basic-research perspective and with the forethought of how such findings may translate with tangible effects in the clinical.

5. Expert opinion

As a leading cause of global morbidity, significant focus is currently being placed on guiding the early detection and diagnosis of cancer by organizations such as the World Health Organization (https://www.who.int/news-room/fact-sheets/detail/cancer). To support such aims, great strides have been collectively made over the last 3 years in the areas of developing the technology (and hardware) that permits the detection of cancers and their there effective treatments, as reliable predictors for patient prognosis.

Against this scientific backdrop, we have focused on the CtsB protease, regulation, biological effects, and as a family member of proteins that are responsible for cancer development and growth, metastasis, chemoresistance, or tumor vascularization. Over the last 20 years, huge developments have been reported in areas spanning molecular mechanisms and rational drug design, with the potential to exploit such findings for diagnostic and prognostic purposes as a foresight. As we report herein, such progress has been impressive and has developed harmoniously, with tangible outcomes that arise from bringing closer inputs from basic research and their clinical applications. Here, the true benefits of such outcomes will arise from the speed at which accurate diagnoses can be made, whether such reagents have the power to resolve the stage of cancer development and the speed with which cancer patients can be treated effectively and monitored in real time. As the CtsB targeting-paradigm outlines herein, such benefits are realistic aims and are achievable, as demonstrated through the development and clinical testing of specific CtsB-specific Prodrugs. In doing so, it defines a ‘molecular framework’ with ‘work flows’ that can be adopted by- or applied to- other cathepsin members with emerging pathological importance. Consequently, future developments of this area do hold much promise as we sit at a very exciting juncture in time, where findings from model systems are beginning to be expeditiously tested within the clinic.

Article highlights

• We introduce the cathepsin proteases and the significance of their expression in cancer

• Cathepsin B is described in the context of its subcellular compartmentalization and contribution to cancer progression

• We then proceed to describe its substrate specificity and how this is modulated by cystatin inhibitors

• We extend such findings to describe the development of small-molecule inhibitors and Activity-Based Probes

• We develop the significance of these to incorporate how such approaches have been adapted to develop cathepsin Prodrugs and their testing in the clinic

• Looking forward, we conclude that cathepsin B inhibition does offer a robust paradigm that will serve to develop novel diagnostic and therapeutic agents through targeting other cathepsin members

This box summarizes key points contained in the article.

Declaration of interests

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

AA Zamyatnin was funded by the Russian Science Foundation (#21-75-30020). LC Gregory, PA Townsend, and SM Soond were funded by the University of Surrey and the University of Surrey Health Partners. This article is dedicated to the brilliant-minded Ms Olga Luzhnowa, 27-12-2022.