Ex vivo fibrosis research: 5 mm closer to human studies
Bigaeva, Emiliia
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“To raise new questions, new possibilities, to regard old problems from a new angle, requires creative imagination and marks real advance in science”. Albert Einstein (1879-1955) The past 40 years of tremendous research efforts have significantly advanced our understanding of the intricate cellular and molecular mechanisms of fibrosis, a pathology with devastating consequences. Fibrosis affects nearly all tissues and organ systems and ultimately results in organ failure. Fibrotic disorders are associated with high morbidity and mortality making them a global health problem. For example, liver fibrosis progresses to cirrhosis and then liver cancer, which is now one of the leading causes of cancer deaths worldwide: with > 90% mortality rate, it takes 750,000 lives per year [1]. The incidence and prevalence rates of other fibrotic diseases are no less overwhelming. Despite the scientific progress in the field of fibrosis research, the clinical success rate in antifibrotic drug development is very limited. This raises the question of how well basic research can be translated to the therapeutic approaches for human disease when relying on conventional preclinical tools. The development and improvement of functional three-dimensional (3D) organ models broadens the possibilities within biomedical research by providing in vitro/ex vivo test settings with high predictive value and relevance to the in vivo environment, and, in case of human-based models, high clinical relevance.
EMERGING 3D TISSUE ENGINEERED MODELS – NOT QUITE THERE YET
Recent advances in cell biology and tissue engineering as well as an increased ethical pressure to develop alternatives to animal models have prompted the development of a wide range of 3D cell and tissue culture technologies [2]. The 3D multicellular organotypic structures not only provide a more in vivo-like context than traditional 2D cell cultures, but also eliminate species differences by allowing drug testing directly in human tissues. Therefore, such models promise to facilitate the early drug development process, starting from disease modelling to target identification and validation, drug screening, efficacy and safety assessment. The emerging 3D bioengineered systems include spheroids, organoids, organs-on-chips, 3D bioprinting and decellularized organs, each with its own advantages and disadvantages.
Spheroids are spontaneous non-adherent aggregations of cells that form a 3D tissue construct.
The cell-to-cell contact in a 3D spheroid creates a native extracellular matrix niche for the cells and maintains their natural environment [3]. Multicellular spheroid cultures were initially developed to recapitulate the functional phenotype of human tumor cells, however, their application extended to many other cell types, including stem cells, hepatocytes and neuronal cells [4]. Recently, vascularized cardiac spheroids have been established by co-culturing cardiac myocytes, endothelial cells and cardiac fibroblasts [5]. These cardiac spheroids responded to transforming growth factor beta (TGFβ) stimulation by increased expression of connective tissue growth factor (CTGF) and fibronectin, and excessive collagen deposition, demonstrating the potential of this model to study mechanisms of
cardiac fibrosis and investigate novel therapeutics in vitro. Furthermore, tumor spheroid monocultures or co-cultures with immune or endothelial cells have been adapted to experimental cancer research and oncology drug screening [6]. In turn, cultivation of primary human hepatocytes in 3D spheroid configuration has been shown to mimic human liver function [7], and when exposed to lipogenic substrates, hepatic spheroids mimicked the pathological condition of hepatic steatosis [8]. While this 3D culture technique is highly reproducible and offers a possibility for high-throughput screening, spheroids fail to recapitulate the structural organization of human organs or maintain physiologically relevant interactions with the extracellular matrix (ECM). They also face several practical challenges such as development and maintenance of spheroids of uniform size, the formation of spheroids from a small seed number of cells, and the precise control of specific ratios of different cell types in a spheroid when in co-culture [4].
Organoids represent dish-based 3D developing tissues that can be defined as “a collection of
organ-specific cell types that develops from stem cells or organ progenitors and self-organizes through cell sorting and spatially restricted lineage commitment in a manner similar to in vivo” [9]. Compared to scaffold-free spheroids, organoids have a higher order self-assembly, resemble organ structure more closely and depend on the matrix (scaffold) for their formation. Organoids are generated from either embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) or primary stem cells, such as tissue-resident adult stem cells [10]. To date, several organoid cultures have been established to resemble various tissues, including pancreas, liver, stomach, intestine, kidney, lung, thyroid, cerebral cortex and retina [4,11]. Human hepatic organoids have been shown to maintain metabolic activity and mimic drug-induced liver fibrosis, while displaying differential toxicity and hepatic stellate cell activation profiles in response to allyl alcohol and acetaminophen [12]. In turn, kidney organoids generated from human iPSCs have been used for toxicity screening in response to cisplatin [13]. Human intestinal organoids (HIOs) established from iPSCs contain both epithelium and mesenchyme, including myofibroblasts - key effector cells in fibrosis [14,15]. It has been shown that HIOs respond to a profibrotic stimulus with TGFβ similar to isolated human myofibroblasts, as well as to the treatment with spironolactone (an aldosterone receptor antagonist that is commonly used as an antifibrotic medication in heart patients), indicating that HIOs mimic intestinal fibrosis and can be used as a screening tool for antifibrotic drugs [16]. Of note, other study showed that spironolactone might exert intestinal toxicity in vitro/in vivo [17]. Beyond such organoids, tumoroids derived from patient cancer tissues provide advanced 3D culture platforms for personalized drug evaluation and development [18,19]. However, few challenges still remain: organoids do not fully recapitulate the structure or function of human organs, since all organoids lack vasculature and interactions with immune cells [9]. Moreover, some organoid cultures replicate only the early stages of organ development, and the characteristics of scaffolds that are used to form the organoids can greatly influence cell adhesion, proliferation, activation, and differentiation [20,21]. Nevertheless, organoids derived from human cells have the potential to provide near-physiological models to study human development and human diseases.
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An organ-on-a-chip refers to an artificial, miniature model of a human organ on a microfluidic cell culture chip [22]. The chips are made with great precision using microfabrication technique and contain continuously perfused chambers inhabited by living cells. Such microfluidic devices contain hollow microchannels to support laminar fluid flow, simulating tissue- and organ-level physiology. The fluidic control also enables delivery of nutrients, growth factors, drugs, or even toxins to the cells grown in the microfluidic channels in a highly regulated spatiotemporal manner [22]. To date, a wide range of organ-on-chip systems have been reported, including skin, lung, liver, kidney, intestine, heart, vasculature and several others [4]. Liver-on-a-chip systems, for instance, can be applied for elucidating drug toxicity and drug testing [23,24]. Lee et al. [25] generated a reversible- and irreversible-injured alcoholic liver disease model in spheroid-based microfluidic chips, where rat primary hepatocytes and hepatic stellate cells (HSCs) are co-cultured. Recently, first NAFLD-on-a-chip system has been established by culturing human hepatoma HepG2 cells in a microfluidic device under free fatty acid supplementation [26]. In turn, kidney-on-a-chip technology has been used for drug-induced nephrotoxicity screening [27]. Advances in the field of microengineering allowed the construction of multi-organ chips, thus enabling interorgan crosstalk. Interaction between intestine and liver co-cultured in a chip was shown for nanoparticle uptake and toxicity [28]. Furthermore, Maschmeyer
et al. [29] developed a four-organ-chip that contains the cell culture equivalent of human intestine,
liver, skin and kidney, connected to each other in a dynamic manner. To our knowledge, such systems haven’t been implemented in fibrosis research yet. However, several technical and biological aspects, such as medium composition, flow or exchange rates, oxygen levels, and waste disposal need to be considered when such multi-organ system are developed [30]. In addition, high costs of the current chip models and their complexity limit their use in a high-throughput setting [31].
3D bioprinting refers to the layer-by-layer printing of living cells, biocompatible materials, and
supporting components to form complex 3D organ-like structures in a highly automated and spatio-temporally controlled manner [32]. 3D bioprinting has been used to generate functional tissues, such as skin, lung, liver, kidney, heart and muscle [2,33]. There is growing evidence for the applicability of bioprinted tissues in drug testing, most of which have been liver models. For example, bioprinted human liver tissue comprising patient-derived hepatocytes, stellate cells and endothelial cells was used to study the hepatotoxic effects of the antibiotics levofloxacin and trovafloxacin [34]. In addition, the bioprinted liver successfully recapitulated basic fibrogenic features following treatment with prototype fibrogenic agents, such as methotrexate and thioacetamide [35,36]. Bioprinting has several advantages, including custom-made microarchitecture and co-culture ability [32]. However, it is still an evolving technology that must overcome several limitations before it can be widely employed. One such limitation is the insufficient resolution with which currently available bioprinters are able to spatially position cells, as compared to the composition found in native organs [37]. Another challenge is the material used to print the cells in: it needs to be compatible with the cells and the printing process, and provide the mechanical and functional properties that resemble human tissues [37]. The emerging alternative approach is scaffold-free bioprinting based on naturally occurring cell-cell interactions, while avoiding the complexity of biomaterials such as biocompatibility, degradation
or potential immunogenicity [38,39]. A common limitation of this approach is that although cells can be manipulated individually, they do not easily form stable assemblies by simply bringing and maintaining them in contact, and this method largely relies on cells to produce the structural cohesion, as their own secreted ECM [40]. Similar to organoids, vascularization of the printed tissues remains challenging, therefore printing living pieces of tissue larger than 1 mm thick is still impossible [41]. Currently, 3D bioprinting requires a complex experimental setup, therefore, limiting its wide use in laboratories.
Recently, the concept of decellularized organs (also referred to as acellular) has emerged as a novel system to study ECM interactions in the context of normal and diseased tissue repair and regeneration [42]. The technique of whole organ decellularization allows the production of 3D organ bioscaffolds while preserving the intrinsic vascular network, bypassing some of the major limitations of other tissue engineered models. Acellular scaffolds are generated by perfusion of decellularization solutions (such as detergents, DNAse and RNAse) through the vasculature [42]. Once decellularized, scaffolds can be recellularized with either iPSCs, primary stem cells or immortalized cell lines. In this case, native ECM guides the cells and aids not only in cell attachment, but also in the formation of various organotypic structures [43]. It has been shown that decellularized healthy lungs, lungs derived from scleroderma, idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) patients maintain both composition and structure of their respective disease pathologies for up to 1 month [44]. Primary human fibroblasts seeded on acellular lung scaffolds derived from patients with IPF were found to obtain a myofibroblast phenotype, as assessed by α-smooth muscle actin (α-SMA) expression. Successful decellularization of whole human liver has also been reported [45], however, such decellularized livers have not been used to test antifibrotic compounds in a multicellular setup [30]. Common problems of this 3D model include the availability of normal human tissues and organs to generate acellular ECM, and the precise cell positioning during recellularization [2]. Most studies recellularize human ECM scaffolds with only few cell types; therefore, they do not fully replicate complex cell-cell interactions.
The field of tissue engineering is moving forward fast, opening a multitude of new possibilities for basic research and drug development that traditional in vitro systems are unable to provide. However, the majority of the aforementioned 3D models commonly face some critical challenges such as integration of vascular and neural networks, maintaining the physiological ratio of different cell types, inclusion of the immune system, restoring regenerative capacity and enabling inter-tissue crosstalk. A number of other things has to be considered. An essential part of any tissue engineered technology is the cell source. While many models rely on cell lines that certainly have an advantage of tractability, low costs and availability, the use of primary cells is imperative when aiming for human-based models in preclinical pharmacological research [2]. The isolation methods of primary cells are improving; however, the use of ESCs is limited due to ethical barriers, and iPSCs as well as mesenchymal stem cells (MSCs) might possess tumorigenicity [46]. Another aspect to consider is that 3D tissue and organ models are far less developed with respect to imaging, analysis, quantification, and automation
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compared with established 2D methods. Furthermore, the application of 3D organ models, such as spheroids and organoids, is well established in toxicological screening and basic research [47]. On the other hand, only a few systems have successfully been implemented in fibrosis research. Most published studies merely challenge a 3D system with profibrotic stimuli, and only a few explore its predictive value for antifibrotic drug efficacy.
HUMAN PRECISION-CUT TISSUE SLICES (PCTS) – FILLING THE GAPS
As an alternative to tissue assembly of individually isolated cells into organized structures, another available method involves the ex vivo use of excised human organs and tissues. These isolated organs may prove extremely valuable for preclinical research as well as for the validation of 3D tissue engineered organ models, serving as an original human reference for tissue structure, cellular composition and (patho)physiological responses [2]. In contrast to the use of whole isolated organs that require complex equipment (e.g. for organ perfusion) and extensively trained personnel, human
precision-cut tissue slices (PCTS) cultivated ex vivo may hold greater potential for preclinical
applications due to their tractability and easier accessibility. PCTS are the best representation of the human organ, with all the cell types present in their original ratio and configuration, and cell-cell and cell-ECM interactions preserved. Moreover, tissue slices retain their native vascular network as well as resident immune cells – both are a major advantage over tissue engineered models. The PCTS culture technique is simple and reproducible.
PCTS cultures have been established from various solid human organs (liver, kidney, heart) as well as non-solid organs (intestine and lung) [48–52]. Human explanted tissue from patients at various disease stages and aetiology (e.g. IPF lungs, cirrhotic livers, kidneys from end-stage renal disease (ESRD) patients or ileum from Crohn’s patients) can be used for obtaining PCTS, which in turn, could be employed to elucidate disease-specific mechanisms of disease pathogenesis and progression. Furthermore, PCTS derived from diseased tissue could serve as a reference for developing new disease models using PCTS from healthy tissues. In this case, healthy slices can be used to study the de
novo disease processes by, for example, challenging PCTS with disease-relevant stimuli. For instance,
treating liver PCTS with high concentrations of lipids and sugars could model non-alcoholic liver disease (NAFLD), whilst culturing liver PCTS with ethanol or alcohol metabolites could be a strategy to model alcoholic liver disease (ALD) [53,54]. Although NAFLD and ALD have been induced in rodent liver PCTS, modelling these pathologies in human PCTS is within our reach, as reported by Palma et
al. [55].
PCTS have long been proven useful in toxicological and pharmacological drug screening [56–63]. As this thesis delineates, PCTS also have the power to advance the field of fibrosis research. The ex vivo PCTS culture is becoming an invaluable tool in unravelling mechanisms of organ fibrosis, as well as in antifibrotic drug testing, as demonstrated by the research in this thesis and by many other studies [64–71]. The unique versatility of this model is reflected by the fact that PCTS develop spontaneous
inflammatory and fibrogenic responses during culture, and each of these responses can be further accentuated by various stimuli (e.g. TGFβ; platelet derived growth factor, PDGF; lipopolysaccharide, LPS).
PCTS in cancer research, personalized medicine and biomarker discovery
As mentioned above, the use of human tissue to prepare PCTS has high clinical relevance, as it allows to eliminate inter-species differences and, more importantly, enables direct therapeutic target validation and testing of individual patient responses to a specific drug (considering that each set of organ slices originates from an individual donor). Over time, data can be accumulated from a large pool of individuals reflecting both human genetic diversity and patterns of disease. Among all possible applications of PCTS, their use in cancer research is gaining more and more attention. Currently, 2D monocultures, tumor xenografts and chemically-induced animal models of cancer are primarily used to identify and screen therapeutic anticancer strategies. It has been suggested that human liver PCTS co-cultured with liver cancer cell lines or patient-derived cancer cells could represent an improved system to study tumor growth and test therapies within the context of a 3D liver environment [72]. A more direct, and perhaps more clinically relevant, approach is to prepare PCTS from tumors. Recently, Misra et al. [73] established and functionally validated the ex vivo organotypic culture of human pancreatic ductal adenocarcinoma (PDAC) PCTS. In particular, these PDAC slices maintained their structural integrity, phenotypic characteristics and functional activity in a 4-day culture. Moreover, the slices were responsive to treatment with rapamycin (an mTOR inhibitor), illustrating the use of PCTS for predicting patient-specific responses and resistance to anticancer agents [73]. The latter is of great relevance to the emerging field of personalized medicine, where PCTS might take a place at the front line. Similar possibilities have been explored with ex vivo cultures of head and neck squamous cell carcinoma and colorectal cancer [74,75]. Besides antineoplastic drugs, oncolytic viruses have been successfully tested in human liver PCTS derived from hepatocellular carcinomas (HCCs) [76]. Lastly, the use of human liver PCTS co-cultured with matched peripheral leukocytes may hold the key for assessing cancer-immune cell interactions, which is of great value for liver cancer research [55].
Accurate disease detection and prognosis, successful clinical drug development and application of personalized medicine – all depend upon the discovery and development of biomarkers. The use of PCTS, especially human tissue-derived slices, may facilitate the development of biomarkers for clinical testing and validation. As part of the (human) PCTS culture routine, media samples can be collected daily, raising the possibility to detect and monitor clinically relevant soluble biomarkers as indicators of disease progression and drug efficacy. This approach was applied for ex vivo biomarker identification for acute drug-induced liver injury [77], and idiosyncratic drug-induced liver injury [78]. Similarly, osteoprotegerin (OPG) has been investigated as a potential biomarker for organ fibrosis in supernatants of murine and human PCTS [79]. In Chapter 4, we performed an initial assessment of selected cytokines secreted by human PCTS derived from healthy and diseased tissues. Although we did not aim to identify new fibrosis biomarkers, we showed that the disease stage of PCTS affects cytokine release. Furthermore, we detected high amounts of secreted tissue inhibitor of metalloproteinase
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(TIMP)-1, metalloproteinase (MMP)-2, osteopontin, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-8 and serum amyloid A (SAA), all of which were previously implicated in fibrosis and/ or suggested as potential serological biomarkers for certain fibrotic diseases and cancers [80–86]. Another approach for the development of biomarkers is the use of transcriptomic profiling of PCTS, which can be further strengthened by the addition of proteomic analysis. In this regard, numerous studies are demonstrating the value of transcriptomics as a clinical tool in the development of biomarkers for a variety of disease states [87]. While the identification of single genes and proteins as potential biomarkers is widely practiced, there is an emerging concept of using a panel of genes that comprise predictive signatures of a disease, detected by transcriptomic analysis. So-called molecular signatures have been successfully identified in oncology [88–92]. One remarkable example is the study of van t’ Veer et al. [88] that reported a molecular signature that predicted the clinical outcome of breast cancer patients with an accuracy that exceeded clinical or histological criteria. Chapters 3 and 4 may hold the key to the identification of such molecular signatures in fibrosis, as transcriptomic profiling of PCTS revealed sets of genes specific to species (mouse and human), organ (liver, kidney, gut) and disease state. Of note, while murine PCTS may not be highly relevant for biomarker discovery, they can be used for identification and validation of preclinical markers. Overall, PCTS can serve as a valuable tool for biomarker discovery and preclinical validation.
Compliance with the 3Rs
The principle of the 3Rs – Replacement, Reduction, and Refinement – originally proposed by Russell and Burch in 1959 [93], embodies the ethical concerns in animal testing and provides a framework for humane research. Replacement encourages the use of alternatives to animal testing, and while all emerging human-based models (3D tissue engineered and human PCTS) fully comply with this principle, the contribution of PCTS to the 3Rs goes beyond that. The number of animals used per experiment can be considerably reduced when working with PCTS, since multiple slices from each organ can be prepared (e.g. ~60 slices can be obtained from mouse liver), allowing to test several experimental conditions at once. Furthermore, several organs from the same animal can be simultaneously sliced, as for example, demonstrated in Chapter 2, where we prepared PCTS from liver, kidney and intestine of the same mouse as one biological replicate. Refinement can be achieved in drug screening studies: the animals do not have to be exposed in vivo to the test compound, and therefore, will not suffer from any possible toxic effects, since PCTS allow for ex vivo drug testing. Therefore, the discomfort of the animals is reduced to a minimum.
Although the 3Rs are now widely recognized by institutional, national and international committees, we, as a society, still need to overcome cultural and historical reliance on animal models. A recent survey (2018) showed that despite the increasing use of human tissue models, the data generated with their use is no more likely to be included in regulatory applications than 4 years ago [94]. The data from human tissue models is often perceived as a distraction from the required animal data, and it may raise additional questions that could impact the success of a submission. Furthermore, some are concerned that results from human-based models will conflict with animal data, invalidating
past experiments [94]. Therefore, to take full advantage of the opportunities offered by human-based models (or any alternative model), continued scientific investment into these models as well as an open dialogue between scientists, pharmaceutical companies and regulators are needed.
LIMITATIONS OF THE PCTS MODEL
As with any model system, there are also limitations inherent to the PCTS method. The foremost disadvantage of the PCTS model as an alternative to animals, is the limited availability of freshly resected human tissue. Both ethical and legal issues associated with the use of human tissue for research purposes have to be considered. Furthermore, some logistical aspects have to be addressed. First, the procedure of acquiring surgical waste material requires coordination and good communication between researchers, surgeons, nurses and clinical pathologists. Second, the time from the moment of resection to the start of human tissue experiment has to be minimized to a few hours. Although the recent study of Bai et al. [95] demonstrated that cryopreservation of lung slices is possible without major impairment of cell viability or tissue functionality, methods of (cryo)preservation still require further improvement for other tissue/organ types [96,97]. The issues related to human tissue are certainly the limiting factor in the extensive use of human PCTS in the industrial settings.
Another limitation of human PCTS is the fact that “healthy” tissue, when taken from resected material or organ rejected for the transplantation, is not truly healthy, although it comes from an area as distant as possible from the diseased area. This increases the variability between subjects and experiments. The same holds true for the use of diseased (fibrotic) slices, as these patients most likely have previously undergone multiple therapeutic strategies prior to surgery, representing another source of variation within the human donors. However, despite the fact that high variation poses a great challenge in any experiment, it reflects the heterogeneity of the human population, capturing the genetic diversity and any other differences (e.g. medical history) that individuals may have. Therefore, one can see it as a strength of PCTS, considering that a similar heterogeneity will eventually appear in clinical trials.
One of the commonly named limitations of the PCTS model is the lack of infiltrating immune cells, which can modulate the disease process. A possible solution would be to culture healthy and diseased slices in combination with immune cells (or subsets of immune cells) to recreate the immunological interactions during the fibrogenic process in (human) tissue. Nevertheless, by not reflecting systemic body responses such as immune cell recruitment or hormonal effects, PCTS enable researchers to investigate local responses.
Lastly, the PCTS model does not allow for direct assessment of inter-organ cross-talk, and its use is currently restricted by the relatively short viability of the slices (48-72h, compared to, for example, several weeks of spheroid culture). Similar to organ-on-a-chip technologies, it is possible to combine different organ PCTS together in one microfluidic device to study organ interactions (e.g. gut-liver axis, liver-kidney axis). For instance, van Midwoud et al. [98] integrated liver and intestinal slices,
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derived from the same rat, into interconnected perfusion chambers in order to recreate intestine-liver cross-talk. Furthermore, implementation of tissue slices into microfluidic systems might improve tissue nutrition and culture duration. Enabling long-term PCTS culture can greatly widen the range of applications in predicting long-term drug toxicity and efficacy. In this thesis we explored another way to improve ex vivo tissue longevity – optimization of culture conditions by enriching the culture media with the growth factors necessary to support cell function, as it was already applied in long-term cultures of liver PCTS [99,100]. In Chapter 7 we showed that the composition of the culture medium has a great influence on the maintenance of intestinal PCTS viability and morphological integrity. Moreover, we demonstrated the importance of medium selection for intestinal slices originating from different species. For instance, medium enriched with noggin, R-spondin and epithelial growth factor (EGF) markedly improved the viability of mouse intestinal PCTS, whereas an addition of Wnt was required to achieve a positive effect on ATP content in rat slices. The differences in Wnt signalling has been previously reported between murine small intestine and colon [101,102]; therefore, it would be interesting to investigate the nuances of Wnt signalling in rat (and human) intestine. The next step would be to assess how various intestinal cell types are maintained during PCTS culture and whether enriched media can improve their survival during prolonged culture.
Overcoming the aforementioned limitations will be the first step on the way to empower PCTS use in preclinical research.
THE MYSTERY OF TISSUE SLICES IN CULTURE
The PCTS technique has been the focus of researchers ever since the Krumdieck slicer was invented in 1980 [103]. However, despite the long history of PCTS and their extensive applications, the recognition of this model has been limited due to the lack of comprehensive molecular characterization and validation. Although numerous studies reported that culture induces inflammation- and fibrosis-like changes in tissue slices, supporting the use of PCTS as a promising ex vivo fibrosis model, these studies often describe only one type of organ PCTS prepared from one species. Therefore, as another empowering step, we investigated how murine and human slices generated from various organs respond to culture in order to get a better understanding of the processes that take place in the PCTS system.
In Chapter 2, we used the concept of conserved, or “core”, mechanisms and pathways of fibrosis that, as now widely accepted, are shared between different tissues and organs. We showed that culturing indeed triggers common mechanisms of fibrogenesis, such as ECM remodelling, and activates TGFβ signalling, a core fibrosis pathway, in murine PCTS, regardless of the organ of origin. Furthermore, we for the first time demonstrated that liver, kidney and intestinal PCTS display substantial differences in the ECM homeostasis prior to culture (e.g. 70% of ECM-related genes were differentially expressed across the organs), respond to culture and involve TGFβ signalling cascades (canonical and non-canonical) in an organ-dependent manner. As a consequence, liver, kidney and intestinal tissues do not have similar susceptibility to antifibrotic treatment with the TGFβ receptor I kinase inhibitor galunisertib.
On a bigger scale, PCTS cultures, as a fibrosis model, not only develop culture-induced inflammatory and fibrogenic responses, but in doing so, they reflect organ-specific features of fibrogenesis.
We continued unravelling the molecular mechanisms involved in PCTS culture by performing whole transcriptome sequencing. Gene expression, being at the core of biological function, is commonly used to investigate cell responses to change and stimuli. One of the advantages of transcriptional profiling on a genome-wide scale is that it does not require prior knowledge of the expression profile of a given cell or tissue, or a pre-existing hypothesis about the gene expression profile typical for a given biological phenomenon [104]. In Chapter 3, we provided in-depth characterization of murine and human PCTS generated from healthy organs, followed by Chapter 4, where we characterized human PCTS from diseased tissues and compared their transcriptional profiles with healthy human PCTS. We discovered the following patterns. First and foremost, we observed that culturing impacts all types of PCTS (i.e., regardless of species, organ or diseased state) in a universal way, by actively inducing inflammatory responses and fibrosis-associated ECM remodelling. We identified IL-11, MMP3 and MMP10 as common modulators of culture-induced dynamic changes across PCTS, as well as commonly activated biological pathways characterized by inflammation and tissue remodelling, such as IL-8 signalling, osteoarthritis pathway and integrin signalling. Second, extensive transcriptional changes in PCTS were not only driven by incubation time, but also by organ type and species of origin. And third, despite the converging effects of culture, diseased phenotype of human PCTS still impacted the biological processes in the tissue (e.g. cytokine release), highlighting the importance of diseased, patient-derived PCTS for preclinical research.
The data acquired by whole-transcriptome sequencing is overwhelmingly extensive, and it can be used in many different ways, far beyond what we could cover in Chapters 3 and 4. For instance, as we often rely on mouse models to predict responses in humans, the transcriptomic analysis gives us an opportunity to assess how similar the molecular processes are in mouse and human PCTS. By taking a closer look at the Venn diagrams in Chapter 3, we can estimate that on average, less than 30% of studied human genes were regulated in a similar way in mouse PCTS for each organ type. This observation is in line with the literature. The conservation of promoters and transcription factor binding sites are important predictors of gene expression similarity across the species [105]. It has been reported that transcription factor binding sites are conserved in hepatocytes for about 30% of the cases when comparing human and mice [106]. Another study showed that there is a 12–18% overlap in differentially expressed genes when in vitro activation of mouse and human hepatic stellate cells was compared [107]. The resemblance of murine and human PCTS in culture can also be assessed on a pathway level. We estimated that on average, 45% of biological pathways that were significantly regulated during culture in human PCTS, were similarly regulated in mouse PCTS (except for colon slices, where the overlap in culture-affected pathways was less than 10%). It is important to be aware of these species-differences and carefully investigate which biological processes can be accurately translated from mice to humans. The overlapping and unique gene and pathway regulation profiles can also be determined for healthy and diseased human PCTS.
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Furthermore, comprehensive transcriptomic analysis could establish the relevance of specific molecular targets uncovered in animal models to human disease. For example, a recent study in rat liver slices established a panel of 12 genes (Col1a1, Has2, Crlf1, Igfbp5, Mmp7, Pappa, Fap, Postn,
Ptx3, Tnc, Tnfsf11 and Wisp1) as the efficacy end-points to validate the antifibrotic effect of tested
compounds [108]. The relevance of these 12 rat genes to human liver can easily be determined by using the provided sequencing data in human PCTS. If we cross-reference the proposed panel of genes with the lists of genes differentially expressed in human PCTS during culture (i.e., supplementary file 1 in Chapters 3 and 4), we would find that only 6 of 12 genes (Has2, Crlf1, Mmp7, Pappa, Fap and
Tnc) might be used as efficacy end-points in human healthy and diseased PCTS. By using the same
supplementary files, one can determine, for example, which transcripts are upregulated in the kidney across two species, including PCTS derived from diseased tissue (e.g. we identified 332 genes, keeping in mind 1:1 mouse-to-human homology), or which transcripts are upregulated across all organs of the same species (e.g. 258 genes are commonly upregulated in human liver, kidney and ileum PCTS during culture), or across all organs of both species.
Another way to use the transcriptomic data presented in Chapters 3 and 4 is to investigate organ-, species- and disease-specific regulators and processes in PCTS, before and after culture. This could be particularly valuable knowledge for the identification of novel therapeutic targets and biomarkers. Overall, generated data from comprehensive gene profiling provides an almost infinite source for basic research, model validation, translatability and drug screening. Future characterization studies of the PCTS culture system will greatly accelerate implementation of this unique and versatile ex vivo model in basic sciences and preclinical drug development.
PCTS: ACCURATE FORECASTING
One of the crucial characteristics of any research model is its predictive power. Although numerous studies showed that PCTS are suitable for antifibrotic drug screening, only a few addressed how well PCTS can predict drug efficacy in vivo or possibly even in the clinic. Therefore, the second part of this thesis was dedicated to answering this question.
Chapter 5 explores the impact of nintedanib, a tyrosine kinase receptor (RTKs) antagonist, on the
progression of renal fibrosis. While other tyrosine kinase inhibitors have already been tested in models of renal fibrosis, we demonstrated that this therapeutic strategy can be evaluated in human PCTS prepared from healthy and diseased kidney tissue. We showed that nintedanib inhibited culture-induced phosphorylation of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) receptors, attenuating fibrosis in human kidney PCTS. These observations largely contribute to the validation of PDGF and VEGF receptors (i.e., RTKs) as clinically relevant therapeutic targets in renal fibrosis. Furthermore, we characterized the phenotype of fibrotic kidney PCTS and demonstrated that the antifibrotic potency of nintedanib diminishes when it comes to the reversal of established fibrosis, which reflects a major challenge in the treatment of end-stage renal disease.
In Chapter 6, we used a panel of three compounds with established antifibrotic activity to pursue a more comprehensive comparison of the data obtained with kidney PCTS with publicly available in vivo data. We demonstrated that changes in key fibrosis end-points evaluated in animal models following treatment (such as attenuation of collagen type I expression and interstitial accumulation, inhibition of TGFβ signalling and/or PDGF signalling and anti-inflammatory properties) were largely detected in murine PCTS, showcasing the high predictive value of slices. Among the three tested compounds, galunisertib showed the most compelling ex vivo antifibrotic profile; however, until now there have been no studies investigating its effects in renal fibrosis in vivo. Galunisertib has been developed as an anticancer agent, and is currently in phase II HCC trials. Therefore, it’s only logical that the first reports of in vivo antifibrotic activity of galunisertib were in animal models of liver fibrosis – CCl4 [109] and Mdr2 knockout (Mdr2KO) mice [110]. The inhibitory effect of galunisertib on collagen expression and/ or deposition was reported in both studies. In Chapter 2, we showed that galunisertib attenuated culture-induced fibrogenesis and ECM remodelling in healthy mouse liver PCTS. Furthermore, we accumulated evidence that galunisertib is also effective in murine PCTS prepared from fibrotic livers of CCl4, Mdr2KO and non-alcoholic steatohepatitis (NASH) mice. In all cases, galunisertib inhibited the gene expression of collagen type I (Figure 1).
Figure 1. Gene expression of key fibrosis markers in murine fibrotic liver slices treated with 10 µM galunisertib for 48h. Three mouse models of liver fibrosis were used: CCl4, Mdr2 gene knockout and NASH (i.e., mice fed high fat diet for
16 weeks). Data are shown as values relative to the control (untreated slices) and expressed as mean ± standard error of the mean (SEM), n=4-14, *p < 0.05.
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Altogether, we successfully demonstrated that responses seen in murine PCTS highly correlated with the responses observed in animal studies, revealing the predictive power of PCTS. Considering that murine PCTS can predict drug efficacy in vivo, it is likely that human PCTS can be used to foretell drug efficacy in clinical trials. In Chapter 6, the lack of clinical efficacy of pirfenidone in patients with kidney disease was reflected in human kidney PCTS. However, it is quite challenging to establish a direct translational link between the response to treatment in human tissue ex vivo and in patients, as different end-points are evaluated. Further studies testing the boundaries of preclinical assessment with PCTS are warranted.
CONCLUDING REMARKS AND PERSPECTIVES
In this thesis, we characterized the ex vivo PCTS culture system, showed its suitability for preclinical drug screening as a translational model with high predictive power and clinical relevance, and optimized culture conditions to improve tissue longevity. Furthermore, our work emphasized the value and advantages of using human tissues for advancing the field of fibrosis research. These studies lay the foundation for the implementation and future validation of the PCTS model in the field of fibrosis research. As with any emerging technology, continuous effort should be invested in improvement, therefore, we suggest a few lines for future research.
In part I of this thesis we thoroughly characterized the molecular processes that take place in PCTS during culture, broadening our understanding of this ex vivo system. We showed that murine and human PCTS undergo dynamic culture-induced transcriptional changes that largely relate to inflammation and matrix remodelling. While culture triggers common mechanisms of fibrosis in all PCTS, transcriptional regulation of genes and biological pathways is influenced by species, organ type and diseased state of the tissue. To extend the provided data, we suggest that transcriptomic analysis should be performed on PCTS cultured for extended periods of time (e.g. 72h), as longer incubation leads to a more pronounced fibrogenic response [49]. Furthermore, samples from more human donors are needed to better represent the genetic diversity of the population. Murine and human PCTS treated with antifibrotics that target distinct elements of the fibrotic program (i.e., core signalling pathways, ECM remodelling, etc.) should be considered for transcriptomic profiling to elucidate molecular signatures that can predict drug efficacy ex vivo. In addition, since transcripts are translated into proteins before becoming effective in an organism, and many species- and organ-specific factors may amplify the differences during the translational process, future studies should combine the results of transcriptomic analysis with large-scale protein measurements to correlate changes in RNA and protein levels. We believe that this approach will strengthen the position of the PCTS model in preclinical research and open great possibilities for therapeutic target identification and validation, and biomarker discovery.
We have shown that intestinal PCTS develop distinct responses during culture compared to liver and kidney PCTS and are less susceptible to antifibrotic treatment. The transcriptional regulation of central fibrosis markers, such as collagen type I and a-SMA, in intestinal PCTS should be further investigated. One critical point that should be addressed is the need for the refinement of human intestine PCTS preparation and/or culture. According to current protocols for human PCTS, only the mucosa is used for slicing; however, other layers of the intestinal wall may play an important role in inflammatory and fibrogenic processes. Therefore, as a possible solution, co-culture of mucosal slices with slices from the muscle layer could be considered, as slicing all layers of the intestinal wall at once imposes technical problems. Compared to other organs affected by fibrosis, the gut is unique due to the presence of the microbiome that has a profound influence on intestinal homeostasis in health and disease [111]. The gut flora is known to affect the systemic availability of drugs and their toxicity; therefore, alterations in the microbiome due to a disease might also impact the efficacy of therapeutics. Since the gut microbiome in mice is remarkably different from that in humans (e.g. almost 85% of gut microbiome that colonizes in human does not exist in mice) [112], the co-culture of human PCTS with intestinal bacteria will be an exciting new area.
In part II, we showed that the PCTS model is an excellent ex vivo screening platform for antifibrotic therapies. We demonstrated that pharmacological interventions, which in our case targeted core fibrosis pathways such as TGFβ, PDGF and VEGF, display unequal efficacy in murine and human kidney PCTS, raising awareness of poor cross-species translation. While murine PCTS can aid bridging the gap between 2D cell cultures and in vivo animal models, human tissue slices are indispensable, if not imperative, for therapeutic target validation and predicting drug efficacy in patients. Future studies should continue to investigate the translational and predictive capacity of PCTS by addressing the questions whether ex vivo data in murine PCTS correlates with in vivo data, and whether data generated with human PCTS is indicative for clinical drug performance.
Considering that fibrosis is not a monocausal disease and the underlying mechanisms are immensely complex, mono-therapy might be insufficient to completely stop the progression of organ fibrosis. Therefore, a combination therapy for fibrosis is an attractive concept. The simple suggestion would be to target two vital but very different pathways: for example, target the upstream (chronic) inflammation and downstream ECM deposition. Such multi-pharmacological approach might provide benefits far beyond single-drug treatments. Compatibility, safety and efficacy of the drug combinations can be easily assessed using PCTS, offering a new line for ex vivo fibrosis research. The compounds with known mechanisms of action or repurposed therapeutics will fit best with this concept. As a starting point, it would be interesting to test a combination of galunisertib and nintedanib for antifibrotic efficacy in kidney PCTS.
Most current PCTS studies, including those described in this thesis, initiate treatment at the time of fibrosis and inflammation induction by culture, which does not reflect the clinical situation. In clinical practice, therapeutic interventions are frequently initiated during late stages of the disease, when fibrosis already exists. Therefore, a delayed exposure of PCTS to the compound of interest should
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be considered for future studies. For example, a 72-hour experiment could be divided in two steps: induction of fibrogenesis during the first 36h of culture followed by 36h of drug exposure. In case of combination therapy, not only simultaneous but also sequential treatment regimens could be of interest.
Further optimization of the PCTS culture system should be carried out with careful assessment of the needs of various cell types to ensure their survival. Additional modifications of the culture conditions could be introduced in order to adapt the PCTS model for specific research questions (e.g. induction or inhibition of specific biological processes with exogenous agents).
Conclusion
In this thesis, we extensively characterized the ex vivo culture of precision-cut tissue slices and provided solid evidence of their high translational and predictive value, endorsing the use of the PCTS model in the field of organ fibrosis research and drug development. We hope that the presented work will lay a strong foundation for the future studies that will eventually lead to the validation of the PCTS model and accelerate its recognition as an invaluable addition to the toolkit with the label “for advanced preclinical drug discovery”. Since the ultimate goal of biomedical research is to understand and cure human diseases, the importance of reliable human-based models in drug discovery cannot be stressed enough. So here we are, with a human tissue slice at the center of it all, a whole 5 mm closer to human studies.
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