Cancer is one of the leading causes of death worldwide. The tumour extracellular matrix (ECM) has unique features in terms of composition and mechanical properties, resulting in a structurally and chemically different ECM to that of native, healthy tissues. This paper reviews to date the efforts into decellularization of tumours, which in the authors’ view represents a new frontier in the ever evolving field of tumour tissue engineering. An overview of the ECM and its importance in cancer is given, ending with examples of research using decellularized tumours, which has already indicated potential therapeutic targets, unravelled malignancy mechanisms or response to chemotherapy agents. The review highlights that more research is needed in this area, which can answer important questions related to tumour formation and progression to ultimately identify new and effective therapeutic targets. Within the near-future of personalized medicine, this research can create patient-specific tumour models and therapeutic regimes. Keywords Cancer, tumour, extracellular matrix, decellularization, decellularized tumour Date received: 17 January 2022; accepted: 17 March 2022 1 Aragonese Agency for R&D (ARAID) Foundation, Zaragoza, Aragón, Spain 2 Multiscale in Mechanical & Biological Engineering Research Group, Aragón Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragón, Spain 3 Division of Biomaterials & Tissue Engineering, UCL Eastman Dental Institute, University College London, London, UK Corresponding author:Elena García-Gareta, Multiscale in Mechanical & Biological Engineering Research Group, Aragón Institute of Engineering Research (I3A), School of Engineering & Architecture, University of Zaragoza, Zaragoza, Aragón, Spain. Email: garciage@unizar.es1091682 TEJ0010.1177/20417314221091682Journal of Tissue EngineeringGarcía-Gareta et al. review-article 2022 Review 2 Journal of Tissue Engineering components. The ECM of tumours is stiffer than that of healthy tissues and this stiffness increases cancer cells prolif-eration and survival, and induces immunosuppression. The ECM is continuously remodelled, not only in tumours but also in healthy tissues. 5 ECM remodelling is crucial for understanding tumour progression.5 The function of cellular and non-cellular TME components is controlled by tumour cells via complex signalling networks, resulting in tumour formation and growth, deficient response to therapy and chemoresistance. The non-malignant cells in the TME pro-mote tumourigenesis throughout tumour development and metastasis. 6 The attention received by the ECM component of the TME has changed considerably over the last decade as its importance in tumour development, progression and metastasis was discovered. As of today, numerous articles can be found highlighting the tumour ECM and its role in malignancy and response to therapy. 4,5 Tissue engineering is based on understanding tissue formation so new functional tissues can be developed in the laboratory. 7 The golden triad of tissue engineering is based on the combination of scaffolds, cells and molecular cues. 7 A scaffold is used by relevant cells to attach to, migrate through it, and proliferate to form the new tissue, and molecular cues are often needed to direct cell fate. These three components are instrumental in developing functional tissues and organs. Since these are also the basic components of cancer tissues, tissue engineers have endeavoured to produce tissue-engineered tumours in the laboratory in order to comprehend cancer mechanisms and test new therapies. 8–10 Tissue engineers possess an array of materials, namely polymers (both natural and synthetic), ceramics or a combination of them (composites), to develop degradable, functional and/or smart scaffolds to act as an ECM for recreating the TME. 9,10 However, the ECM is a complex entity in terms of both composition and structure that is constantly remodelled by tightly regulated remodelling mechanisms. 3,11 Therefore, many features of the ECM are lost when building scaffolds using the above-mentioned materials. As a result, tissue engineers often use decellularized matrices as scaffolds that offer the advan-tage of great similarity with the native tissue. 3,12 Decellularization is the process of eliminating native cells and genetic material from the ECM while ideally main-taining its structural, biochemical and biomechanical properties. 12 Decellularized tissues can then be repopu- lated with cells of interest.12 The vast majority of the research into tissue-engineered tumour models using decellularized tissues uses native tis-sue, and therefore a normal ECM, that is then repopulated with cancer cells and sometimes other non-cancerous cells associated with the tumour of interest. 3,13–15 However, the tumour ECM has unique features in terms of composition, for example, increased collagen deposition, and mechani-cal properties, that is, increased stiffness due to the heavy crosslinking of abundant collagen, resulting in a structurally and chemically different ECM to that of native tissues. 4,16 Therefore, very recently scientists started inves- tigation into the decellularization of tumours, thereby pro-ducing decellularized tumour ECM (dT-ECM) that not only maintains the mechanical characteristics of the tumour but also retains its composition and structure. The use of these dT-ECM in tissue-engineered tumour models could very closely imitate actual tumours. The aim of this paper is to review to date the efforts into decellularization of tumours, which in the authors’ view represents a new frontier in the ever evolving field of tis-sue engineering and more specifically tumour tissue engi-neering. But first, the ECM and its important role in cancer will be described in order to frame the relevance and importance of decellularizing tumours to unravel key molecular and cellular mechanisms behind cancer. The extracellular matrix and its importance in cancer The extracellular matrix and its continuous remodelling The non-cellular component found in all tissues and organs of our body is the ECM, of which two main forms exist: the interstitial ECM and the basement membrane. 5,11,17,18 The interstitial ECM is a porous and interconnecting three-dimensional (3D) network that surrounds stromal cells and can connect to the basement membrane, which is a stable, sheet-like dense structure that lines the basal surface of, for instance, epithelial and endothelial cells and separates tis-sues into different and well-organized compartments. 5 Apart from acting as a physical scaffold for cells, the ECM pro-vides essential chemical, physical and mechanical signals necessary for tissue morphogenesis, differentiation and homoeostasis. 11,17 These various signals are detected by a multitude of cell surface receptors, thereby triggering intra-cellular signalling cascades that for instance result in the expression of relevant genes that regulate apoptosis, cell proliferation, migration or differentiation. 11,17 Throughout life in both healthy and diseased states like cancer, resident cell types secrete the molecules that make up the ECM, which are modified in response to mechanical cues and stimuli such as oxygen and nutrient availability. 11,19 Fundamentally, the ECM is composed of water, proteins and polysaccharides. 17 The major protein component is the collagen superfamily, which makes it the most abundant family of proteins in the human body. 20 The protein compo- nents of the interstitial ECM are mainly collagen types I, III, V , etc., fibronectin and elastin. 5,18 The basement membrane is mainly composed of collagen type IV , laminins and network-bridging proteins such as nidogen and heparan sul-phate proteoglycans. 5,18 Nevertheless, each tissue and organ possess an ECM with a distinct composition and topology. 20 García-Gareta et al. 3 Different remodelling mechanisms ensure the ECM is continuously changed in terms of structure, composition, abundance and organization of individual components.5,21 Ultimately, changes in the ECM affect the microenviron-ment surrounding cells consequently changing cell behav-iour. ECM remodelling is highly complex with over 700 proteins involved in the various mechanisms, which include: (1) ECM deposition, where the biochemical and mechanical properties of the ECM can be tuned by chang-ing the amount and composition of ECM components; (2) chemical modification at the post-translational level, thereby altering the biochemical and structural characteris-tics of the ECM; (3) proteolytic degradation, where bioac-tive ECM fragments and ECM-bound factors are released; and (4) force-mediated physical remodelling, where the organization of the ECM is changed by aligning ECM fibres and creating new routes for cell migration. 5,21 Tissue homoeostasis depends on ECM remodelling and its tight regulation, as changing single elements of this delicate remodelling balance can have important effects on com-plex cellular signalling networks. This is because the vari-ous ECM components act as ligands for cell surface receptors such as integrins or tyrosine kinases. 22,23 The extracellular matrix in cancer Unsurprisingly, in pathological states like cancer, ECM remodelling is dysregulated. 5 Tumourigenic remodelling of the interstitial ECM in cancer induces a myriad of bio-chemical and biophysical changes that affect ECM stiff-ness, cell signalling and migration and tumour progression. 24 Furthermore, in malignant tumours, inva- sion of stromal tissue by cancer cells is a consequence of the tumourigenic remodelling of the basement mem-brane. 25 Cancer cells have the capacity to alter the four ECM remodelling mechanisms discussed in the previous paragraph. 5 As a result, a cancer-supporting ECM is assembled, which can comprise up to 60% of the tumour mass. The most notable features of tumour ECM are increased collagen deposition leading to an over-abun-dance of collagen, altered specific collagen types ratios and stiffness. 4,26 The tumour ECM strongly differs in amount of deposi- tion, composition, organization and post-translational modification from the ECM in normal tissue. 4 The tumour - igenic ECM is deposited by CAFs as well as the tumour cells themselves. 27,28 Infiltration of fibroblasts and myofi- broblasts and subsequent accumulation of significant amounts of a collagen-rich ECM is seen in many solid tumours. This process is called desmoplasia and is strongly linked to poor prognosis and resistance to systemic ther - apy. 29–31 CAFs contribute to a more malignant tumour phe- notype by driving epithelial-to-mesenchymal transition (EMT) and induce production of collagen and other ECM molecules by supporting tumour cells via paracrine stromal cell-derived factor-1 (SDF1) and transforming growth factor beta (TGFβ) signals. 32–34 Accumulating evi- dence in the literature shows that platelets play a key role in promoting tumour growth and metastasis through inter - action with tumour cells at multiple levels. 35,36 The ability of inducing platelet aggregation by many cells present in solid tumours correlates with metastatic potential. 35 Tumour cell-activated platelets secrete multiple factors that in turn target not only tumour cells but also others pre-sent in the TME thus affecting the tumour ECM. 35 Tumour- cell activated platelets crucially contribute to the establishment and expansion of the early metastatic niche and various mechanisms towards this effect have been pro-posed by researchers. 36 Briefly, the three most well-studied mechanisms are: (1) preparation of the initial metastatic microenvironment by forming the ECM and recruiting granulocytes, (2) creation of a neovasculature and (3) crea-tion of an immune-suppressive environment around the developing metastases. 36 Several ECM molecules such as fibrillar collagens, fibronectin, elastin and laminins are expressed in high lev-els in many solid tumours. 37,38 Some cancers are particu- larly rich in hyaluronan, for instance pancreatic ductal adenocarcinomas. 39 In comparison to benign mammary lesions, production of collagen is shifted towards collagen types I and III mainly in the stromal part of the tumour in invasive ductal carcinomas. 40,41 Since the benign lesions also consist mainly of fibroblastic cells, this manifests the tendency and capacity of breast tumour cells to change the secreted matrisome of its stroma. Furthermore, in the des-moplastic stroma of breast carcinomas, up to 15% of the collagen component of the matrix consists of collagen type V , which has low abundance in normal and fibrocystic (<0.1%) breast tissue. 42 It has been shown that increasing the collagen V/I ratio reduces length and organization of collagen fibres to the point that fibre formation can be inhibited, resulting in a gel-like ECM. 43,44 Another exam- ple is ovarian carcinomas, where compared to benign tis-sue, collagen type IV is downregulated and its expression is inversely correlated with stage and markers of malig-nancy. 45 Increased collagen type I expression in melanoma is observed and corresponds with invasiveness, angiogen-esis and reduced survival. 46,47 Finally, a fast collagen turn- over in tumours is evidenced by high collagen mRNA expression, the aberrant form of fibrous collagen spindles and the increased expression of matrix proteases. 41 The stiffness of the tumour ECM differs from normal ECM.26,48 Tumour ECM stiffness is a consequence of tumour growth, and is related to an increase in metastasis rate and poor clinical outcomes. 49 Furthermore, tumour ECM stiffness disrupts tissue morphogenesis by increas-ing cell tension. 50 The increased stiffness in the tumour ECM results from an aggregation of ECM proteins that enclose packs of hyaluronic acid gel-like structures, which make tumours resistant to compressive stresses. In the 4 Journal of Tissue Engineering periphery, collagen and fibronectin are accumulated.51 The increased collagen content and crosslinking are both cor - related to an enhancement of ECM stiffness and cancer malignancy.52 Regarding the increased degree of collagen crosslinking observed in tumour ECM, lysyl oxidase (LOX) is an amine oxidase responsible for collagen cross-linking and its amount is frequently elevated in cancers. 50 Increased matrix crosslinking forces tumour progression by providing enhanced integrin signalling. ECM stiffness may be involved in the rise of tumour incidence with age-ing, as aged tissues are stiffer and contain high amounts of aberrant collagen crosslinking. 50 As we have seen, the tumour ECM significantly differs from that in normal tissue: in a nutshell, the tumour ECM is more abundant, denser and stiffer. These altered features can negatively affect response to therapy. 4 For instance, an excessive accumulation of dense and rigid ECM, encapsu-lating clusters of tumour cells, can act as a barrier, thereby shielding the cells from therapeutic agents. Furthermore, this barrier also impairs diffusion of oxygen, nutrients and metabolites, leading to increased hypoxia and metabolic stress which activate anti-apoptotic and drug resistance pathways. Chemoresistance is also a consequence of cell–ECM contacts and increased tissue stiffness via integrin and FAK-signalling. 4 This section merely offers an overview of the ECM and its alteration in cancer, which as the reader can gather, is a highly complex matter. If the reader wishes to learn more about the subject, there are numerous and excellent reviews which dwell on multiple or specific aspects and concepts discussed above. 4,5,16,26,35,36,51 Our aim was simply to high- light the importance of the ECM in cancer to set the tone for the following sections. Decellularization of tumours Rationale and importance As mentioned in the Introduction, cancer is one of the big- gest health challenges of our time, and tissue-engineered tumour models can help not only to comprehend cancer formation and progression mechanisms, but also to iden-tify and test new therapies. The vast majority of tissue-engineered tumour models uses either a scaffold made of a biodegradable material, or decellularized healthy ECM, that are then populated with cancer cells. However, as seen in the previous section, the tumour ECM considerably dif-fers from the ECM found in healthy tissues and therefore, models that use decellularized healthy ECM are missing important features of the ECM component of the TME. For instance, fibrillar collagens, fibronectin, elastin, and laminins are expressed in high levels in many solid tumours compared to the expression levels observed in non-tumoural tissues. Therefore, these expression levels will be missed in decellularized healthy ECM. Moreover, the increased levels of collagen, which as described in the pre-vious section is heavily crosslinked, confer a stiffness to the tumoural ECM which will be absent from the decellu-larized healthy ECM. Consequently, producing dT-ECM for its use in tissue-engineered tumour models could very closely imitate actual tumours, which adds a level of mim-icry absent from tissue-engineered models that use decel-lularized healthy ECM or fabricated scaffolds. We believe this added level of mimicry is needed to answer important questions about tumour formation and progression and identify new therapeutic targets. A simple search on PubMed of ‘decellularized tumour’ rendered 251 results since 2010, out of which only 49 arti-cles actually reported on dT-ECM (Figure 1). The rest of the articles were either reviews, or studies where decellu-larized healthy ECM was used to create tissue-engineered tumour models. As it can be seen, there is a positive trend in the number of publications that use decellularized mate-rials for tissue-engineered tumour models. However, only a small fraction of them actually used decellularized tumours, for example, in the last year 2021, 50 publica-tions reported on the use of decellularized materials for tissue-engineered tumour models, out of which 35 used healthy ECMs, 5 were reviews, and only 10 reported on dT- ECM. Since the tumour ECM is considerably different to native ECM, decellularized tumours could be a power - ful tool in the study of cancer and new therapies for the reasons already exposed in this review. Before producing them, it is important to consider the tissue source, the decellularization method, the characterization of the dT-ECM and its recellularization with relevant cell types. Tissue source Tumour tissue can be obtained from different sources, namely human or animal derived, and cell culture derived. Table 1 shows the different options as well as their advan-tages and disadvantages. Tumours isolated from the human body have a tumour ECM that is truthful to the real-life situation. On the other hand, ethical approval and patient consent can sometimes be tedious to obtain, which delays and constrains tissue collection for experimentation. Besides, tissue availability is limited when isolating tumours from human patients, which hampers large scale in vitro studies. Patient varia-bility is also another disadvantage of using human-derived tumour ECM: even for the same cancer type, the ECM will vary between different patients. Some of the disadvantages of using human-derived tis- sue can be solved with xenotransplantation models. In these models, human cells, tissues or organs can be implanted into an immunodeficient animal host, which provides the environment for the human tissue to function and grow normally. 71 These models have been studied for organ transplantation or toxicity studies,71,72 and have García-Gareta et al. 5 emerged as a powerful research tool in the fight against cancer.53,54 With these models, it is possible to grow an unlimited number of patient-specific tumours that can then be harvested for experimentation, including decellulariza-tion studies. Alternatively, cancer cell lines can also be used to induce the tumours. 54–58 For instance, Wishart and colleagues55 used the epithelial, human breast cancer cell line MDA-MB-231 to create a xenotransplantation model of breast cancer. MDA-MB-231 cells were injected into the mammary gland of immunocompromised mice and were left to grow for 8 weeks. Parallel to this, PyMT-MMT mice (mammary tumour transgenic mouse model carrying the polyomavirus middle T antigen oncogene) were kept for 10 weeks, as this is a mouse model of breast cancer that Figure 1. PubMed search for the phrase ‘decellularized tumour’ showing results from 2010 until 2021. Results are broken down into articles reporting into actual dT-ECM (decellularized tumour ECM), reviews, and the rest (studies where decellularized healthy ECM was used for tissue-engineered tumour models). Table 1. Summary of tumour tissue sources discussed in the text. Origin Source Advantages Disadvantages Human derived53–61Tumours isolated from human patients- Have a tumour ECM that is truthful to the real-life situation- Procurement of ethical approval and patient consent - Tissue availability- Patient variability Tumours isolated from xenotransplantation models- Have a tumour ECM that is truthful to the real-life situation - Eliminates issues of patient variability and tissue availability- Procurement of ethical approval and patient consent - Need dedicated facilities for animal work - Animal work is costly Animal derived 62,63Animal models of cancer- Eliminates subject variability- Eliminates issue of tissue availability- The tumour ECM will differ from that found in human tumours - Procurement of ethical approval- Need dedicated facilities for animal work- Animal work is costlyInduced - Eliminates subject variability- Eliminates issue of tissue availability - Can isolate tumours at different stages of malignancy Cell culture derived 64–70Cultured primary cancer cells- Eliminates issue of tissue availability - In the case of primary cancer cells, the deposited tumour ECM will resemble that found in patients in terms of composition - In the case of cancer cell lines, subject variability is eliminated - Cost-effective- The tumour ECM will differ from that found in human tumours - The tumour ECM can be easily altered by the culture conditions- Procurement of ethical approval to use primary cancer cellsCultured cancer cell lines 6 Journal of Tissue Engineering grows tumours in all mammary glands that closely resem- ble the progression and morphology of human breast can-cers. 62 The authors reported that at the 8 and 10 weeks’ time points all tumours had metastases to the lungs. The tumours were extracted and successfully decellularized using a detergent-based protocol (Figure 2(a)–(e)). They were then seeded with MDA-MB-231 cells that had been labelled with green fluorescent protein (GFP) and live imaged for 16 h, showing that ECM isolated from xeno- graft 231-tumours significantly increased cell migration and proliferation (Figure 2(f)–(h)). Seeded GFP-labelled PyMT tumour–derived cells in the ECM isolated from pri-mary MMTV-PyMT tumours increased tumour cell inva-sion without affecting proliferation (Figure 2(f)–(j)). The authors concluded that whole-tissue ECM can be isolated from healthy and tumour-containing mammary glands and used to study ECM-driven effects on individual cell phenotypes. 55 Animal derived tumour tissue is also a relevant source for decellularization procedures. Animal models of cancer exist, like the just mentioned PyMT-MMT mice model of breast cancer. 62 The option of inducing a tumour is also Figure 2. (a) Generation of tumours with MDA-MB-231 xenografts (231-tumour) and tumours from PyMT-MMTV (PyMT- tumour) mice. (b) Study design. (c) Haematoxylin and eosin (H&E) staining of mammary gland, 231-tumours and PyMT-tumours before and after decellularization (scale bar = 200 μm). (d) Representative western blot of 231-tumour before and after decellularization for cellular compartments. (e) Photo before and after decellularization of PyMT-tumour. (f) Representative tracks (each line is a cell over 16 h) of 231-GFP or PyMT-GFP cells seeded on decellularized ECM from mammary gland (Mg), 231-tumours or PyMT-tumour. (g) Percentage of proliferation for 231-GFP cells on healthy mammary gland or 231-tumour. (h) Cell migration speed of 231-GFP seeded on decellularized ECM from mammary gland and 231-tumours. (i) Percentage of proliferation for PyMT-GFP cells on mammary gland or PyMT-tumour scaffolds. (j) Cell migration speed of PyMT-GFP seeded on decellularized ECM from mammary gland and PyMT-tumours. For proliferation, data point is a field of view. For cell migration, each point is the average speed of a cell over 16 h. Data shows mean ± SEM from at least three decellularized ECM scaffolds from three mice. Statistical significance was analysed by unpaired two-tailed Mann Whitney t test. **p < 0.01 and ***p < 0.005. ns, not significant. Image from Wishart et al. 2020 55 (open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited). García-Gareta et al. 7 possible. For example, Saldin and colleagues63 induced oesophageal adenocarcinoma in Sprague-Dawley rats by subjecting them to the Levrat surgical procedure. In this procedure, the jejunum is anastomosed to the distal oesophagus, thereby creating constant acid reflux and chronic inflammation in the distal oesophagus. Over the next 17–33 weeks, normal oesophageal squamous epithe- lium transforms to metaplastic Barrett’s oesophagus (i.e. intestinal metaplasia) and then to a neoplastic, glandular cell type. These changes in cell phenotype mimic the pathophysiology of gastroesophageal reflux disease. The advantage of this procedure is that the authors could har - vest the tumour tissue at different stages of the disease. 63 Some of the disadvantages of using tumour tissue isolated from human patients, for example, patient-specificity and tissue availability, are solved by using animal derived tumour tissue as a source. Nevertheless, ethical constraints are still a disadvantage of this tumour tissue source. Furthermore, dedicated facilities for animal work are needed, which are not always easily accessible. This is also the case with xenotransplantation models. In cell culture derived tumours, cells (primary or cancer cell lines) are cultured using standard mammalian tissue culture techniques, and the tumour ECM deposited by the cells over time is then decellularized and used for investi-gation. 64–68 Cell culture derived tumour ECM offers evi- dent advantages such as plenty availability and cost-effectiveness. Moreover, in the case of primary can-cer cells, the deposited tumour ECM will resemble that found in patients in terms of composition, whilst in the case of cancer cell lines, subject variability is eliminated. Nevertheless, the deposited ECM will differ from that found in real tumours, especially in the case of tumour ECM derived from cultured cell cancer lines. However, this is an important source of tumour ECM for experimen-tation as evidenced by the literature. For instance, Wang and colleagues 69 investigated the underlying mechanisms behind the acquisition of resistance to EGFR (epidermal growth factor receptor) tyrosine kinase inhibitors (TKIs), which remains a critical problem in lung cancer. Although it is known that the TKI-induced or -selected genetic changes drive resistance, this also occurs without genetic changes in tumour cells. The authors showed that the ECM derived from various components of the TME may drive resistance in the absence of genetic changes: culturing tumour cells that were otherwise sensitive to EGFR TKIs on dT-ECM, or co-culturing with the tumour ECM donor cells, immediately conferred resistance to them. Furthermore, they showed that the primary constituent in the tumour ECM driving resistance may be collagen, partly through the collagen receptor Integrin-β1. The effect of the tumour ECM and collagen was dose-dependent and reversible, thus revealing a potential therapeutic target. The dT-ECM used to reach these relevant conclusions was derived from the culture of lung cancer cell lines and fibroblasts. 69 A last and interesting example is the work by Nayak and colleagues.70 In this study, the authors engi- neered a biochemico- and mechano-mimetic 3D culture platform for primary breast cancer cells by decellularizing CAFs cultured on 3D macroporous polycaprolactone (PCL). Briefly, the authors cultured CAFs for 7 days on 3D porous PCL scaffolds that had an elastic modulus (4.0 ± 0.5 kPa) comparable to that of breast tumours (4.04 ± 0.93 kPa). Decellularization included various methods such as freeze-thaw and detergents. As a result, the decellularized scaffolds had a PCL backbone, which mimicked the mechanical properties of breast tumours, coated with a natural tumour ECM, which mimicked the biochemical properties of metastatic breast tissue. 70 Other studies beyond decellularization have also developed tis-sue-engineered tumour models based on the culture of can-cer cell lines, like the work by Martinez and co-workers where the luciferase-tagged SKOV-3 ovarian cancer cell line, the CAFs cell line human ovarian cancer fibroblast IHFOT-208 and the OVCAR-8 cell line were used to cre-ate a preclinical 3D ovarian cancer model using a perfu-sion bioreactor. 73 A last example is the paper by Stamati and colleagues74 were CAKI-2 and 786-O RCC renal cell carcinoma lines were used to create biomimetic 3D renal tumoroids to study the effect of the tyrosine kinase inhibi-tor Pazopanib on killing cancer cells and disruption of endothelial networks. Decellularization methods, characterization and recellularization As of today, the same methods used in the decellulariza- tion of tissues and organs are used to decellularize tumours. 3,12 These include biological, chemical or physical methods.12,75 Within each method category, different agents and techniques exist. Usually, a combination of methods is used (Table 2). Biological decellularization methods include the use of enzymes to break down DNA or RNA fragments (nucleases), cell-matrix interactions (trypsin) or target peptide bonds (pepsin). They are normally used with other chemical agents like surfactants, which disarrange the phospholipid cell membrane. For instance, D’Angelo and colleagues 77 used a surfactant-enzymatic treatment on both healthy liver and colorectal metastatic liver and reported a phenotypical appearance similar to the non-decellularized tumours. Other chemical methods like hypertonic and hypotonic solutions, which induce an osmotic shock, have also been used in the decellulariza-tion of tumours alongside other methods and agents. As an example, Pinto and co-workers 78 used a decellulariza- tion method on colorectal specimens, both tumour and normal, that included a first step using a hypotonic buffer, followed by a 24 h treatment with 0.1% SDS, three washes with the hypotonic buffer, and a 8 Journal of Tissue Engineering Table 2. Summary of decellularization methods used to decellularize tissues including tumours. Method Agent (examples)/technique Description Comments Biological3,12,75–78Enzymes (nucleases, trypsin, pepsin)- Nucleases break down DNA or RNA fragments. - Trypsin breaks down cell-matrix adhesions. - Pepsin targets peptide bonds.- Ineffective on their own so they are used in combination with other methods and agents. Chemical 3,12,75–78Surfactants (sodium dodecyl sulphate (SDS), sodium deoxycholate (SD),Triton X-100, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulphonate (CHAPS))- Surfactants disarrange the phospholipid cell membrane, thereby lysing cells. - They can be ionic, non-ionic or zwitterionic.- SDS is effective at removing cells and genetic material but it is cytotoxic, thus it needs extensive washing. - SD is less damaging and cytotoxic than SDS. - Non-ionic surfactants like Triton X-100 are less damaging to tissue structure than ionic surfactants like SDS and SD. - They can cause DNA agglutination. - Surfactants tend to be used in combination with other methods and agents. Acids and bases(alkaline treatment, peracetic acid, organic acids)- Acids and bases solubilize the cell membrane and nuclear material due to their intrinsic charge properties.- Peracetic acid is highly corrosive and has a strongly oxidising nature. - Peracetic acid increases ECM stiffness. - Used in combination with other methods and agents. Hypotonic and hypertonic solutions- These solutions use osmotic properties to make the cells explode.- The osmotic shock kills the cells, but it does not remove the cell waste that it releases to the matrix. Physical 3,12,75,76,79–88Agitation/stirring/rotation/shaking- Commonly used to facilitate chemical agent infiltration and to induce cell lysis.- As a facilitator, it needs to be used in combination with other methods. - An aggressive agitation or rotation can greatly damage the ECM. Freeze-thaw - Freezing temperatures are alternated with biological temperatures for an optimized number of cycles, thereby lysing cells.- Ineffective at removing cells and genetic material. - It is used in combination with other methods and agents. High hydrostatic pressure - Applying pressures above 600 MPa to dismantle the cellular membrane.- It can be used on its own.- Excessive pressure can damage the structure. Supercritical CO 2- Supercritical fluids have liquid-like density and gas-like diffusivity. - With a critical temperature of 31.1°C and a critical pressure of 7.40 MPa, supercritical CO 2 is compatible with biological systems.- CO2 does not remain within the tissue and therefore, extensive washing is not required. - CO2 is non-polar, so a polar entrainer is necessary to remove the polar phospholipid membrane. - It can be used on its own. Sonication - Sonication is the process of applying sound energy to agitate samples in a liquid. - Ultrasonic frequencies (>20 kHz) are usually used. - Enables chemical agents to reach the more inner parts of the tissue.- As a facilitator, it needs to be used in combination with other methods. - An aggressive sonication process can greatly damage the ECM. Vacuum-assisted decellularization- Enables chemical agents to reach the more inner parts of the tissue.- As a facilitator, it needs to be used in combination with other methods. García-Gareta et al. 9 3 h digestion with DNase. The authors performed the protocol under constant agitation to aid the infiltration of the different agents into the samples. It was reported that no visible nuclei could be observed in normal and tumour matrices after decellularization. DNA quantification confirmed the efficient removal of about 99.3% of the total DNA in normal tissue and 99.6% in tumours. Haematoxylin and Eosin (H&E) and Masson’s trichrome staining confirmed the absence of cell remnants in both normal and tumour decellularized tissues with preserva-tion of a collagen-rich ECM network. 78 The study by Pinto et al. evidences that some physical methods like agitation, rotation, shaking or sonication can facilitate the decellularization process. 83–86 Freeze-thaw is another physical method that has been used in the decellulariza-tion of tumours. The work by Romero-López and col-leagues 87 is an example, where an initial freeze-thaw cycle was included in the decellularization protocol, fol-lowed by surfactant treatment and extensive washing to eliminate the detergents. The process was aided by the use of stirring. Other promising and efficient physical methods such as high hydrostatic pressure or supercriti-cal CO 2, which yield excellent results in normal tis- sues,79–82,88 have not yet been used in the decellularization of tumours to the best of the authors’ knowledge.Characterization of the dT-ECM follows the decellulari- zation process. As with decellularized healthy tissues, the main objective is to confirm elimination of cellular material and preservation of the original ECM, including composi-tion, structure and mechanical properties. 12 Table 3 sum- marizes available methods commonly used to achieve this objective. Typically, the native or non-decellularized tissue is characterized alongside the decellularized one for com-parison and assessment of efficiency. Macroscopically, the decellularization process can be followed by observing the change in colour and light trans-mittance from the native tissue (pink to orange/reddish and opaque) to the decellularized one (white and translucent) (Figure 3). Histological and immunohistological stainings are popular and somewhat unavoidable methods for assessing decellularization. Not only they detect specific components and structures but also some of them can qual-itatively confirm elimination of cellular material, that is, H&E and Masson’s trichrome stainings. DAPI (4′,6-diami-din-2-fenilindolo) or Hoechst (bis-benzimides) stainings can also be used to qualitatively confirm elimination of cellular material as they bind to AT-rich regions of dsDNA (double stranded DNA). Nevertheless, quantitative confir - mation can be obtained by quantifying the DNA content. As an example the work by Lü and colleagues, 56 where Table 3. Summary of common methods used in the characterization of decellularized ECM, either obtained from healthy tissue or tumours. Purpose Characterization method Comments Elimination of cellular materialH&E staining Stains cell nuclei purple/blue and the ECM pink DAPI or Hoechst stainings Bind to AT-rich regions of dsDNA Masson’s trichrome staining Stains cell cytoplasm red and collagens blue Quantification of DNA content Quantifies the amount of DNA present in the ECM Preservation of the original ECMImmunohistochemical staining of specific ECM components, e.g. laminin, fibronectin, collagens type I and IVDetects the pattern of expression of specific ECM components Alcian blue or Toluidine blue stainings Stain glycosaminoglycans (GAGs) blue Periodic acid-Schiff staining Detects polysaccharides like glycogen, and mucosubstances such as glycoproteins, glycolipids and mucins in tissues Silver stain Detects reticular collagen fibres Quantification of collagen content Quantifies the total amount of collagen present in the ECM Quantification of GAGs content Quantifies the total amount of GAGs present in the ECM Proteomics Characterizes the proteome, that is, the entire set of proteins present in the ECM. Fourier transform infrared spectroscopy (FTIR)Characterizes the infrared spectrum of the ECM Scanning electron microscopy (SEM) Characterizes the architectural features of the ECM, e.g. pores, fibres Quantification of architectural parameters with an image analysis softwareCharacterizes percentage of porosity, pore size range or fibre diameter Mechanical testing Characterizes mechanical properties of interest, which include tensile strength, elastic modulus, stiffness or yield strength Rheology Characterizes the viscoelastic properties of the ECM Table compiled from