3D In Vitro Model (R)evolution: Unveiling Tumor–Stroma Interactions

Complex tumor–stroma interactions, a key feature of most solid tumors, drive tumor progression, metastasis, and drug resistance, and ultimately lead to treatment failure.Clinically relevant 3D in vitro models are necessary to recapitulate the complex interactions between tumor and stromal cells, and provide tools to better understand the molecular mechanisms as well as for testing anticancer therapies.Novel bioengineered models, organoid systems, and microfabrication technologies, such as 3D bioprinting, have delivered key innovations towards more advanced platforms for tumor modeling in vitro. The complex microenvironment in which malignant tumor cells grow is crucial for cancer progression. The physical and biochemical characteristics of this niche are involved in controlling cancer cell differentiation, proliferation, invasion, and metastasis. It is therefore essential to understand how cancer cells interact and communicate with their surrounding tissue – the so-called tumor stroma – and how this interplay regulates disease progression. To mimic the tumor microenvironment (TME), 3D in vitro models are widely used because they can incorporate different patient-derived tissues/cells and allow longitudinal readouts, thus permitting deeper understanding of cell interactions. These models are therefore excellent tools to bridge the gap between oversimplified 2D systems and unrepresentative animal models. We present an overview of state-of-the-art 3D models for studying tumor–stroma interactions, with a focus on understanding why the TME is a key target in cancer therapy. The complex microenvironment in which malignant tumor cells grow is crucial for cancer progression. The physical and biochemical characteristics of this niche are involved in controlling cancer cell differentiation, proliferation, invasion, and metastasis. It is therefore essential to understand how cancer cells interact and communicate with their surrounding tissue – the so-called tumor stroma – and how this interplay regulates disease progression. To mimic the tumor microenvironment (TME), 3D in vitro models are widely used because they can incorporate different patient-derived tissues/cells and allow longitudinal readouts, thus permitting deeper understanding of cell interactions. These models are therefore excellent tools to bridge the gap between oversimplified 2D systems and unrepresentative animal models. We present an overview of state-of-the-art 3D models for studying tumor–stroma interactions, with a focus on understanding why the TME is a key target in cancer therapy. The composition of the tumor microenvironment (TME, see Glossary) and tumor stromal interactions are major factors that aggravate tumor growth and metastasis, leading to poor clinical outcomes [1.Avnet S. et al.Pre-clinical models for studying the interaction between mesenchymal stromal cells and cancer cells and the induction of stemness.Front. Oncol. 2019; 9: 305Crossref PubMed Scopus (9) Google Scholar, 2.Mitchell M.J. et al.Engineering and physical sciences in oncology: challenges and opportunities.Nat. Rev. Cancer. 2017; 17: 659-675Crossref PubMed Scopus (132) Google Scholar, 3.Asghar W. et al.Engineering cancer microenvironments for in vitro 3-D tumor models.Mater. Today. 2015; 18: 539-553Crossref Scopus (168) Google Scholar, 4.Langhans S.A. Three-dimensional in vitro cell culture models in drug discovery and drug repositioning.Front. 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Cancer. 2017; 17: 659-675Crossref PubMed Scopus (132) Google Scholar], including cancer-associated fibroblasts (CAFs), endothelial cells, pericytes, immune cells [such as lymphocytes, neutrophils, dendritic cells (DCs), and monocytes] that are the most prevalent cell types (Figure 1). Other less prevalent factors include myeloid-derived suppressor cells (MDSCs) and mesenchymal stromal cells (MSCs) [7.Maman S. Witz I.P. A history of exploring cancer in context.Nat. Rev. Cancer. 2018; 18: 359-376Crossref PubMed Scopus (144) Google Scholar], as well as platelets [9.De Palma M. et al.Microenvironmental regulation of tumour angiogenesis.Nat. Rev. Cancer. 2017; 17: 457-474Crossref PubMed Scopus (627) Google Scholar,11.Yan M.J. Jurasz P. The role of platelets in the tumor microenvironment: from solid tumors to leukemia.Biochim. Biophys. Acta, Mol. Cell Res. 2016; 1863: 392-400Crossref PubMed Scopus (90) Google Scholar]. These stromal cells actively interact with tumor cells, among themselves, and with the ECM by secreting chemokines, growth factors (GFs), enzymes, extracellular vesicles, and miRNAs that regulate the expression of genes and proteins which influence metabolic pathways related to cancer [12.Ramamonjisoa N. Ackerstaff E. Characterization of the tumor microenvironment and tumor–stroma interaction by non-invasive preclinical imaging.Front. Oncol. 2017; 7: 28-37Crossref PubMed Scopus (45) Google Scholar]. As such, some cell types can either promote or suppress tumor growth depending upon the cellular context [13.Bellomo C. et al.Transforming growth factor β as regulator of cancer stemness and metastasis.Br. J. Cancer. 2016; 115: 761-769Crossref PubMed Scopus (109) Google Scholar]. Hence, much focus has been on accurately modeling TME interactions in vitro and in vivo. In the field of cancer research, especially for testing anticancer drugs, many experiments are still performed with 2D cocultures, xenografts, or syngeneic mouse models. Nevertheless, 2D models are too simple and are unable to mimic the complexity and dynamic interactions of the TME. Cells grow on a flat plastic surface as a monolayer, and this can result in loss of crucial cellular signaling pathways and changes in cell responses to stimuli [14.Duval K. et al.Modeling physiological events in 2D vs. 3D cell culture.Physiology. 2017; 32: 266-277Crossref PubMed Scopus (478) Google Scholar, 15.Mabry K.M. et al.Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype.Biomaterials. 2016; 74: 31-41Crossref PubMed Scopus (57) Google Scholar, 16.Melissaridou S. et al.The effect of 2D and 3D cell cultures on treatment response, EMT profile and stem cell features in head and neck cancer.Cancer Cell Int. 2019; 19: 1-10Crossref PubMed Scopus (58) Google Scholar]. Moreover, 2D cultures do not conserve the original shape and polarization of cells (Table 1). By contrast, animal models are usually too expensive, complex, difficult to work with, and are associated with ethical problems. It is also challenging to analyze some effects that too often are not representative of human-specific events, which limits the applicability of these models [3.Asghar W. et al.Engineering cancer microenvironments for in vitro 3-D tumor models.Mater. Today. 2015; 18: 539-553Crossref Scopus (168) Google Scholar,17.Sung K.E. Beebe D.J. Microfluidic 3D models of cancer.Adv. Drug Deliv. Rev. 2014; 79: 68-78Crossref PubMed Scopus (107) Google Scholar]. By contrast, multicellular 3D in vitro systems can overcome these limitations and bridge the gap between experimental tractability and physiological relevance. 3D models can reproduce mechanical and biochemical cues that are crucial for cancer development, such as morphology, cell–cell/cell–ECM interactions, tissue stiffness, and specific gradients [4.Langhans S.A. Three-dimensional in vitro cell culture models in drug discovery and drug repositioning.Front. Pharmacol. 2018; 9: 6Crossref PubMed Scopus (370) Google Scholar,14.Duval K. et al.Modeling physiological events in 2D vs. 3D cell culture.Physiology. 2017; 32: 266-277Crossref PubMed Scopus (478) Google Scholar,18.Jensen C. Teng Y. Is it time to start transitioning from 2D to 3D cell culture?.Front. Mol. Biosci. 2020; 7: 33Crossref PubMed Scopus (80) Google Scholar]. However, these models usually only feature specific interactions between one component of the TME and tumor cells. Thus, although new models are being developed, the complexity of the tumor stroma in vitro has not yet been achieved. Nevertheless, recent advances in 3D cancer models have the potential to (i) improve drug discovery, (ii) be used as platforms for drug testing, and (iii) enable the development of personalized cancer treatments [3.Asghar W. et al.Engineering cancer microenvironments for in vitro 3-D tumor models.Mater. Today. 2015; 18: 539-553Crossref Scopus (168) Google Scholar].Table 12D versus 3D Cell Culture MethodsCharacteristics2D3DRefsCell morphologyAltered cell shape, usually flat and elongated; loss of epithelial cell polarityThe natural cell shape and polarization is preserved; cells grow in 3D aggregates[15.Mabry K.M. et al.Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype.Biomaterials. 2016; 74: 31-41Crossref PubMed Scopus (57) Google Scholar]Gene expressionCell adhesion-, proliferation-, and survival-related genes are usually modifiedAccurate representation of gene expression patterns[16.Melissaridou S. et al.The effect of 2D and 3D cell cultures on treatment response, EMT profile and stem cell features in head and neck cancer.Cancer Cell Int. 2019; 19: 1-10Crossref PubMed Scopus (58) Google Scholar,85.Riedl A. et al.Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT–mTOR–S6K signaling and drug responses.J. Cell Sci. 2017; 130: 203-218Crossref PubMed Scopus (184) Google Scholar,86.Luca A.C. et al.Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.PLoS One. 2013; 8e59689Crossref PubMed Scopus (185) Google Scholar]Cell proliferation and differentiationCell differentiation is poor and proliferation occurs at an unnaturally rapid paceCells are well differentiated; proliferation is realistic depending on 3D matrix interactions[15.Mabry K.M. et al.Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype.Biomaterials. 2016; 74: 31-41Crossref PubMed Scopus (57) Google Scholar]Cell interactionsDeprived of cell–cell and cell–ECM interactions, no cell niches are createdCell junctions are common and allow cell communication[14.Duval K. et al.Modeling physiological events in 2D vs. 3D cell culture.Physiology. 2017; 32: 266-277Crossref PubMed Scopus (478) Google Scholar]Tumoral heterogeneityBasic; all cells receive the same amount of nutrients; inaccurate replication of the TMEBetter approximation and representation of the TME; nutrients are not equally supplied[4.Langhans S.A. Three-dimensional in vitro cell culture models in drug discovery and drug repositioning.Front. Pharmacol. 2018; 9: 6Crossref PubMed Scopus (370) Google Scholar,51.Costa E.C. et al.3D tumor spheroids: an overview on the tools and techniques used for their analysis.Biotechnol. Adv. 2016; 34: 1427-1441Crossref PubMed Scopus (216) Google Scholar]Response to stimuliInaccurate representation of mechanical and biochemical cuesCells grow in a 3D environment and receive stimuli from all directions that properly represent in vivo stimuli[16.Melissaridou S. et al.The effect of 2D and 3D cell cultures on treatment response, EMT profile and stem cell features in head and neck cancer.Cancer Cell Int. 2019; 19: 1-10Crossref PubMed Scopus (58) Google Scholar,85.Riedl A. et al.Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT–mTOR–S6K signaling and drug responses.J. Cell Sci. 2017; 130: 203-218Crossref PubMed Scopus (184) Google Scholar]ReproducibilityHighly replicableDifficult to replicate some conditions[4.Langhans S.A. Three-dimensional in vitro cell culture models in drug discovery and drug repositioning.Front. Pharmacol. 2018; 9: 6Crossref PubMed Scopus (370) Google Scholar]Analysis and quantificationEasy interpretation of results; better long-term culturesDifficult to analyze data, especially with multiple cell types or when in spheroid/organoid conformation[4.Langhans S.A. Three-dimensional in vitro cell culture models in drug discovery and drug repositioning.Front. Pharmacol. 2018; 9: 6Crossref PubMed Scopus (370) Google Scholar,17.Sung K.E. Beebe D.J. Microfluidic 3D models of cancer.Adv. Drug Deliv. Rev. 2014; 79: 68-78Crossref PubMed Scopus (107) Google Scholar]CostCheaper for large-scale studiesMore complex and expensive techniques[18.Jensen C. Teng Y. Is it time to start transitioning from 2D to 3D cell culture?.Front. Mol. Biosci. 2020; 7: 33Crossref PubMed Scopus (80) Google Scholar,51.Costa E.C. et al.3D tumor spheroids: an overview on the tools and techniques used for their analysis.Biotechnol. Adv. 2016; 34: 1427-1441Crossref PubMed Scopus (216) Google Scholar] Open table in a new tab This review presents a landscape of current 3D in vitro models for studying the complex interactions within the TME, including cell/ECM-based assays, cell-based models, and microfluidics. The advantages and limitations of each platform are discussed, followed by critical analysis of the factors that novel culture platforms should address to establish more clinically relevant 3D models. ECM proteins are one of the most relevant components of the tumor stroma, and they actively interact with almost all cell types and control their behavior and responses to external stimuli. Dynamic changes in ECM components regulate cell proliferation, migration, adhesion, differentiation, cytoskeletal organization, and cell signaling [4.Langhans S.A. Three-dimensional in vitro cell culture models in drug discovery and drug repositioning.Front. Pharmacol. 2018; 9: 6Crossref PubMed Scopus (370) Google Scholar,19.Bonnans C. et al.Remodelling the extracellular matrix in development and disease.Nat. Rev. Mol. Cell Biol. 2014; 15: 786-801Crossref PubMed Scopus (1636) Google Scholar]. For example, ECM stiffness (desmoplasia), alongside rapid cancer cell proliferation and the establishment of a poor blood vessel network, contributes to cellular hypoxia in epithelial-derived tumors [19.Bonnans C. et al.Remodelling the extracellular matrix in development and disease.Nat. Rev. Mol. Cell Biol. 2014; 15: 786-801Crossref PubMed Scopus (1636) Google Scholar,20.Emon B. et al.Biophysics of tumor microenvironment and cancer metastasis – a mini review.Comput. Struct. Biotechnol. J. 2018; 16: 279-287Crossref PubMed Scopus (83) Google Scholar]. Furthermore, secretion of hypoxia-inducible factors (HIFs) by tumor cells induces macrophage and fibroblast recruitment to hypoxic regions of the primary tumor, leading to increased ECM remodeling and angiogenesis [21.Gilkes D.M. et al.Hypoxia and the extracellular matrix: drivers of tumour metastasis.Nat. Rev. Cancer. 2014; 14: 430-439Crossref PubMed Scopus (657) Google Scholar]. The formation of an abnormal ECM that stimulates cancer progression starts with the activation of CAFs, which contribute to tissue fibrosis and matrix stiffness, by alignment and building up of collagen fibers (mediated primarily by LOX enzymes) [22.Hamidi H. Ivaska J. Every step of the way: integrins in cancer progression and metastasis.Nat. Rev. Cancer. 2018; 18: 533-548Crossref PubMed Scopus (354) Google Scholar]. In addition, deregulated ECM supports epithelial cellular transformation and hyperplasia [19.Bonnans C. et al.Remodelling the extracellular matrix in development and disease.Nat. Rev. Mol. Cell Biol. 2014; 15: 786-801Crossref PubMed Scopus (1636) Google Scholar]. Matrix metalloproteinases (MMPs) are essential for ECM degradation and tumor cell invasion, and also contribute to the formation of metastatic sites and angiogenesis support by the TME [19.Bonnans C. et al.Remodelling the extracellular matrix in development and disease.Nat. Rev. Mol. Cell Biol. 2014; 15: 786-801Crossref PubMed Scopus (1636) Google Scholar,22.Hamidi H. Ivaska J. Every step of the way: integrins in cancer progression and metastasis.Nat. Rev. Cancer. 2018; 18: 533-548Crossref PubMed Scopus (354) Google Scholar]. In addition, integrins are associated with every step of carcinogenesis because they function as signaling receptors that control cellular adhesion, migration, mechanotransduction, and ECM remodeling [22.Hamidi H. Ivaska J. Every step of the way: integrins in cancer progression and metastasis.Nat. Rev. Cancer. 2018; 18: 533-548Crossref PubMed Scopus (354) Google Scholar]. For instance, increased ECM stiffness is correlated with overexpression of β1 integrins in cancer cells, and this induces the activation of focal adhesion kinase and RhoA/Rho-associated protein kinase signaling [22.Hamidi H. Ivaska J. Every step of the way: integrins in cancer progression and metastasis.Nat. Rev. Cancer. 2018; 18: 533-548Crossref PubMed Scopus (354) Google Scholar]. In addition, integrin-mediated alignment of fibronectin fibers within the tumor ECM by CAFs contributes to directional cancer cell migration [23.Erdogan B. et al.Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin.J. Cell Biol. 2017; 216: 3799-3816Crossref PubMed Scopus (179) Google Scholar]. These changes in the composition and architecture of the ECM over time demonstrate the complex spatiotemporal dynamics of the TME that are associated with targets of tumor progression, and can lead to promising pathways for the development of novel and more effective therapies. Given that the ECM is both a major TME component and a tumor-inducing factor, cell/ECM-based assays have been developed to manipulate the interactions between cells and the surrounding matrix in a physiologically relevant setting. An ideal scaffold should provide an appropriate environment for cell adhesion, proliferation/differentiation, and migration to allow the generation of in vitro tumor models that closely recapitulate essential cell–ECM interactions. In this context, tumor cells can be cultured within biomaterials, including decellularized native tissues, or in 3D scaffolds based on ceramics or synthetic and/or natural polymers. Hydrogel-based scaffolds are typically preferred owing to the possibility of tailoring their mechanical properties to closely mimic the tumor ECM (Figure 2). Scaffolds produced from synthetic polymeric biomaterials include polyethylene glycol (PEG), polycaprolactone (PCL), poly(hydroxyethylmethacrylate) (PHEMA), poly(lactic-co-glycolic acid) (PLGA), and ceramics (such as hydroxyapatite or bioglass) [24.Feng S. et al.Expansion of breast cancer stem cells with fibrous scaffolds.Integr. Biol. 2013; 5: 768-777Crossref Scopus (50) Google Scholar,25.Long T.J. et al.Prostate cancer xenografts engineered from 3D precision-porous poly(2-hydroxyethyl methacrylate) hydrogels as models for tumorigenesis and dormancy escape.Biomaterials. 2014; 35: 8164-8174Crossref PubMed Scopus (20) Google Scholar]. Synthetic polymers allow more control over the properties of the scaffold and the ability to modulate them as required. The surface of synthetic polymers can be modified to incorporate peptides, such as RGD (Arg-Gly-Asp) peptides or fibrinogen, that promote protein adsorption and cell adhesion [5.Rodenhizer D. et al.The current landscape of 3D in vitro tumor models: what cancer hallmarks are accessible for drug discovery?.Adv. Healthc. Mater. 2018; 7e1701174Crossref PubMed Scopus (31) Google Scholar]. In addition, hybrid scaffolds combine soft hydrogels with polymeric scaffolds and cells [26.Rijal G. Li W. 3D scaffolds in breast cancer research.Biomaterials. 2016; 81: 135-156Crossref PubMed Scopus (99) Google Scholar]. For instance, PEG heparin hydrogels were used to demonstrate the role of integrins in tumor cell–ECM interactions by culturing breast and prostate cancer cells on hydrogels functionalized with the different peptide motifs RGD, GFOGER (collagen I), or IKVAV (laminin-111) [27.Taubenberger A.V. et al.3D extracellular matrix interactions modulate tumour cell growth, invasion and angiogenesis in engineered tumour microenvironments.Acta Biomater. 2016; 36: 73-85Crossref PubMed Scopus (75) Google Scholar]. Hence, the choice of biomaterials as well as the physical/chemical conditions of the scaffold determine how the cells will react to the substrate and play an important role in the experimental outcome (Figure 2A,B). Natural biomaterials include collagen, fibrin, alginate, and chitosan that can be sourced from tissues and cells [28.Grolman J.M. et al.Rapid 3D extrusion of synthetic tumor microenvironments.Adv. Mater. 2015; 27: 5512-5517Crossref PubMed Scopus (69) Google Scholar, 29.Rebelo S.P. et al.3D-3-culture: a tool to unveil macrophage plasticity in the tumour microenvironment.Biomaterials. 2018; 163: 185-197Crossref PubMed Scopus (66) Google Scholar, 30.Hume R.D. et al.Tumour cell invasiveness and response to chemotherapeutics in adipocyte invested 3D engineered anisotropic collagen scaffolds.Sci. Rep. 2018; 8: 12658Crossref PubMed Scopus (5) Google Scholar]. Alternatively, decellularized ECM (dECM) offers the advantage of recreating natural biochemical environments without compromising the tissue-specific architecture and the ECM, thereby generating scaffolds that have biochemical and structural cues similar to those present in vivo [31.Ferreira L.P. et al.Decellularized extracellular matrix for bioengineering physiomimetic 3D in vitro tumor models.Trends Biotechnol. 2020; 38: 1397-1414Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar,32.Lü W.D. et al.Development of an acellular tumor extracellular matrix as a three-dimensional scaffold for tumor engineering.PLoS One. 2014; 9e103672Crossref PubMed Scopus (54) Google Scholar]. Most commonly used ECM substitutes, such as Matrigel, incorporate undefined and highly variable factors that can affect the experimental results and the reproducibility of the model [33.Drost J. Clevers H. Organoids in cancer research.Nat. Rev. Cancer. 2018; 18: 407-418Crossref PubMed Scopus (400) Google Scholar]. Given the close resemblance to the native matrix structure, cell–ECM interactions can be more easily replicated in dECM-based models after cellularization. In addition, dECMs are promising alternatives to better control the TME in vitro, and have advantages over scaffolds that focus only on individual ECM components and not on the ECM environment as a whole [34.Gill B.J. West J.L. Modeling the tumor extracellular matrix: tissue engineering tools repurposed towards new frontiers in cancer biology.J. Biomech. 2014; 47: 1969-1978Crossref PubMed Scopus (59) Google Scholar]. The decellularization process, however, has its limitations because it is challenging to ensure tissue intactness after treatment with detergents and enzymes. With these considerations in mind, multiple modifications of scaffold-based cell culture supports have been optimized for tumor modeling. A tissue matrix scaffold (TMS) using native ECM has been developed to overcome cell culture methods that do not mimic the biophysical/biochemical properties of the ECM [35.Rijal G. Li W. A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening.Sci. Adv. 2017; 3e1700764Crossref PubMed Scopus (53) Google Scholar]. This in vitro model consists of a multilayered tissue culture platform prepared from decellularized mouse mammary tissue. Cancer and stromal cells are cultured in a compartmental fashion that induces the expression of intracellular and extracellular biomarkers of breast cancer cells, thus confirming correct tumor growth and proliferation. This TMS therefore mimics the structure of the mammary tissue while providing a simple-to-use tool for screening specific tumor biomarkers [35.Rijal G. Li W. A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening.Sci. Adv. 2017; 3e1700764Crossref PubMed Scopus (53) Google Scholar]. In a different approach, a 3D tumor model was developed by generating anisotropic collagen scaffolds seeded with adipocytes and tumor cells [30.Hume R.D. et al.Tumour cell invasiveness and response to chemotherapeutics in adipocyte invested 3D engineered anisotropic collagen scaffolds.Sci. Rep. 2018; 8: 12658Crossref PubMed Scopus (5) Google Scholar]. This model allowed examination of adipocytes in the tumor stroma by culturing breast cancer cells in collagen pores aligned perpendicular to the surface, thus reflecting the in vivo microenvironment in which the ECM organizes in an anisotropic 3D spatial configuration. The invasion of tumor cells into the stroma was compared between two different cell lines overexpressing either Wnt1 or Her2. The presence of adipocytes increased the migration of both cancer cell types, and promoted cancer cell invasion, while reducing the overall number of migratory cells, thus demonstrating the heterogeneity of cellular behavior in this model [30.Hume R.D. et al.Tumour cell invasiveness and response to chemotherapeutics in adipocyte invested 3D engineered anisotropic collagen scaffolds.Sci. Rep. 2018; 8: 12658Crossref PubMed Scopus (5) Google Scholar]. Scaffolds can provide a proper ECM-mimicking environment for culturing cells and have advantages over 2D models and in vivo models. Scaffolds offer an inexpensive and easily analyzable platform which has tunable and instructive properties that can recapitulate relevant biochemical and structural cues [26.Rijal G. Li W. 3D scaffolds in breast cancer research.Biomaterials. 2016; 81: 135-156Crossref PubMed Scopus (99) Google Scholar]. One study showed a significant difference in the levels of gene expression affecting ECM remodeling, namely processes associated with cell adhesion and tumor growth, and this resulted in increased radio- and chemoresistance in the 3D Matrigel model compared with the 2D culture, which highlights the poor reproduction of in vivo findings in 2D models [36.Zschenker O. et al.Genome-wide gene expression analysis in cancer cells reveals 3D growth to affect ECM and processes associated with cell adhesion but not DNA repair.PLoS One. 2012; 7e34279Crossref PubMed Scopus (93) Google Scholar]. Furthermore, scaffolds can be applied in most current 3D in vitro models, and have been used to produce complex 3D bioprinted models, to induce the assembly of cell spheroids, and to promote the 3D culture of cells in microfluidic platforms, as explained in detail in the next sections. The use of scaffolds in the fabrication of 3D in vitro models has paved the way for novel techniques such as 3D bioprinting that use such scaffolds to create more complex models with well-defined architecture, composition, and high reproducibility (Figure 2C) [37.Langer E.M. et al.Modeling tumor phenotypes in vitro with three-dimensional bioprinting.Cell Rep. 2019; 26: 608-623Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. Cell printing is an emerging approach for 3D cancer cell patterning that facilitates the control of spatial and temporal distribution of cells [3.Asghar W. et al.Engineering cancer microenvironments for in vitro 3-D tumor models.Mater. Today. 2015; 18: 539-553Crossref Scopus (168) Google Scholar]. Common 3D bioprinting techniques include extrusion-, inkjet-, and stereolithography-based bioprinting, as well as laser-assisted and electrospinning-based bioprinting (Box 1) [38.Heinrich M.A. et al.3D bioprinting: from benches to translational applications.Small. 2019; 15e1805510Crossref PubMed Scopus (80) Google Scholar]. The process of printing must avoid damaging pressure/heat sensitive fluids, especially when printing living cells [3.Asghar W. et al.Engineering cancer microenvironments for in vitro 3-D tumor models.Mater. Today. 2015; 18: 539-553Crossref Scopus (168) Google Scholar]. Therefore, the choice of biomaterial must consider its biocompatibility, the shape-fidelity of the material, and the level of instructiveness required.Box 1Microfabrication Techniques to Produce 3D Constructs and Microfluidic DevicesOver the past decades, multiple 3D bioprinting methods have been improved to allow the design of complex cell-laden 3D constructs. Each of these technologies is application-specific and the choice depends on the type of bioink used [40.Moroni L. et al.Biofabrication stra