AMR: Active microrheology; AFM: Atomic force microscopy; SHG: Second harmonic generation; n/a: not applicable; HE: Hematoxylin & Eosin; PAS: Periodic acid-Schiff; SEM: Scanning electron microscopy; SDS: sodium dodecyl sulfate; GAG: Glycosaminoglycan; n/e: not evaluated. Overall, decellularization protocols aim to eliminate all cell material while maintaining ECM architecture and biochemical components and are mainly a combination of physical, chemical and enzymatic methodologies ^[41][144]. The removal of cellular components without impairing the original ECM architecture, protein and glycoprotein distribution, and viscoelastic properties renders decellularization a reliable and attractive technique for studying ECM ^{[19][26][36][37]}[19,26,49,52]. Several decellularization protocols and methodologies for evaluating the biochemical and biomechanical features of decellularized CRC ECM have been reported (Table 1). The lack of uniformity in these approaches obviously results in differences in decellularization efficiency, ECM characterization and ECM-mediated biological effects. Therefore, the uniformization of methodologies concerning CRC tissue decellularization would be essential for the reproducibility of results. Decellularized ECM can be cell-, animal-, or human- or patient-derived and have been applied as scaffolds for 3D cell cultures ^[42][145], bioprinting techniques for creating adaptable 3D structures, and as components in synthetic and natural cell culture platforms ^[43][146], providing bioactive ECM components in tunable constructs ^[44][147]. For example, the use of porcine jejunum decellularized matrix, derived from the patented Biological Vascularized Scaffold (DE:302014007893; BioVaSc^®, proprietary knowhow Fraunhofer IGB) ^[45][148], is an interesting 3D in vitro colon model. This scaffold functions as an alternative biological colon-like structure with a mucosal tissue layer, including crypt, villi and a basement membrane, allowing the investigation of tumor cancer cell growth, EMT processes, cell invasion across the basement membrane, and metastization. Importantly, once this dynamic model is maintained in a bioreactor, the repopulation with CRC cells (i.e., SW480) and fibroblasts results in the formation of tumor-like clusters. Such data highlight the relevance of mimicking the tumor milieu to support malignant and stroma cell interactions ^[39][142]. Another exciting CRC biomimetic model was described by Alabi et al., who repurposed the use of mouse decellularized colon matrices, recellularized with HT-29 and HCT-116 human CRC cell lines, to investigate the crosstalk between ECM and cancer cell traits. Interestingly, mouse colon scaffolds were a better support for CRC cell HT-29 proliferation and differentiation than Matrigel^TM. Moreover, HCT-116 cells displayed a higher rate of cell invasion when cultured in mouse colon cancer decellularized matrices than in cells cultured in wild-type mouse colon scaffolds or Matrigel^TM [40][143]. Three-dimensional ECM-hydrogels from decellularized human normal and tumor colon tissues have already been prepared through lyophilization, powdering and solubilization techniques and were useful for showing that tumor ECM components induced faster growth of HT-29 cells and their shift toward a glycolytic metabolism ^[37][52]. In an elegant study by Tian et al., organ-specific metastases were obtained by seeding CRC cells in a biomatrix coating composed of mouse lung and liver decellularized ECM ^[46][149]. In this system, 3D colonies were spontaneously formed and mimicked in vivo metastasis in terms of histological, molecular and phenotypic characteristics. Remarkably, these conditioned cancer cells exhibited tissue-specific tropism when injected into Nu/Nu mice. Despite the undeniable utility and potential of these works that clearly show the impact of the matrix in CRC progression and metastasis, these approaches lack the native ECM architecture and mechanical properties that exhibit an active role in cell behavior ^[7][19][7,19]. In human CRC, strategies using decellularized ECM to study TME dynamics have been broadly focused on patient-derived scaffolds. Several protocols have been developed with the aim of efficiently removing cellular components from intestinal tissue while maintaining the architecture and biomechanical/biochemical features (Figure 2). These scaffolds have been successfully recellularized with different types of cells ^[19][26][36][19,26,49]. In 2016, Chen and Shuler described detailed procedures for establishing an organotypic human colon model from decellularized biopsy specimens, which were then recellularized with primary colon epithelial cells, endothelial cells, and fibroblasts ^[38][141]. This methodology allowed the identification of several genes that drive invasion in APC and KRAS mutated cells and demonstrated this model’s success in studying cancer biology under physiologically relevant conditions that include cell–matrix interactions and the spatial localization of multiple cell types, crucial in the TME context. In this field, approaches that consider paired CRC and normal adjacent tissue benefit from the direct comparison of samples from the same individual and allow the consideration of the role of tumor versus normal ECM on various cancer-associated activities and interactions with other TME components. Nevertheless, studies with access to this type of exceptionally valuable sample have been mainly restricted to recellularization with only one type of cell and require a further complex to create a structure that most trustworthily resembles the TME. Beyond proteomic and structural characterization of the decellularized ECM, reports showed that tumor ECM modulates IL-8 expression by HT29 cells [19] and that both HT-29 and HCT-116 cells reduced the sensitivity to 5-fluoracil when in a 3D ECM-setting ^[36][48][49,53]. In comparison with 2D cultures, HT-29 cells grown in CRC patient-derived scaffolds also displayed changes in the expression of genes and proteins related to proliferation and an increase in those concerning pluripotency and stemness ^[49][151]. From a different perspective, Pinto et al. [26] implemented a novel approach by studying the effect of human decellularized normal and tumor matrices derived from CRC patients’ surgical resections on the macrophage inflammatory signature. This work showed for the first time that, although derived from the same patient, normal and tumor matrices differently modulated the macrophage phenotype, with the last inducing an anti-inflammatory polarization, mimicking the immunosuppressive tumor microenvironment. Additionally, macrophages differentiated within tumor decellularized matrices stimulated CRC cell invasion through the expression of CCL18, an immunosuppressive chemokine identified as a key molecule in this process. Decellularized tissues have also been applied in the study of the CRC metastatic process. D’Angelo and colleagues created a model with decellularized normal and primary tumor CRC tissue, as well as matched CRC liver metastasis with the aim of recapitulating this specific microenvironment in vitro ^[48][53]. This system demonstrated that HT-29 cells cultured in scaffolds derived from liver metastasis exhibit a higher EMT transition, a loss of E-cadherin and a higher vimentin expression, among other biological processes. One of the major drawbacks of CRC patient-derived scaffolds is the limited amount of tumor tissue available from each individual, since it derives from biopsies or surgical resections, from which most tissue is required for further diagnostic molecular and histological characterization. Another question to keep in mind is that normal mucosa adjacent to the tumor, while often considered a healthy control from the same individual, in fact represents an intermediate state between normal and tumor tissues ^[50][152]. Despite collecting from at least 10 cm away from the tumor, a normal adjacent mucosa has a large number of differentially expressed genes in comparison with normal mucosa from healthy donors. This genetic disparity is essentially related to functions concerning the inhibition of matrix metalloproteinases (MMPs), cell adhesion molecules, TGF-β and integrin signaling pathways, inflammation, and cytokine–receptor interaction ^[51][153]. Interestingly, an integrative analysis of TCGA and GTEx RNA-seq data showed that normal adjacent tissue from the sigmoid colon is more similar to the tumor, while normal adjacent tissue from the transverse colon is more comparable to healthy tissue ^[50][152]. The recellularization of decellularized ECM also presents a few challenges, namely the choice of cell(s), the cells’ distribution within the scaffold and the reproducibility of recellularization efficiencies, even in samples from the same patient ^[52][154]. Concerning the specific case of CRC, strategies have been focused on small decellularized tissue fragments, static culture conditions, and recellularization with cancer cell lines. However, more sophisticated systems for mimicking the TME will require the inclusion of immune and stromal cells under dynamic cultures that will allow the flow of nutrients and molecules between the different compartments. To surpass the issue of spatial heterogeneity of the matrisome ^[53][155], tissue samples have to be representative of distinct tumor regions to avoid biasing experimental outcomes. These are relevant topics to be considered when establishing an organotypic 3D model for CRC cancer with decellularized tissues, as well as for previously determining if there was previous neoadjuvant therapy. Still, the possibilities of these kinds of systems to incorporate ECM, cancer, stromal and immune cells will allow the study of the dynamic and complex crosstalks between the different components and recapitulate more closely the TME and, eventually, design strategies with potential for predicting clinical outcomes.