Organoids are simple tissue-engineered cell-based in vitro models that recapitulate many aspects of the complex structure and function of the corresponding in vivo tissue. They can be dissected and interrogated for fundamental mechanistic studies on development, regeneration and repair in human tissues, and can also be used in diagnostics, disease modelling, drug discovery and personalized medicine. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumours. To date, numerous organoid engineering strategies that support organoid culture and growth, proliferation, differentiation and maturation have been reported. This Primer highlights the rationale underlying the selection and development of these materials and methods to control the cellular/tissue niche; and therefore, the structure and function of the engineered organoid. We also discuss key considerations for generating robust organoids, such as those related to cell isolation and seeding, matrix and soluble factor selection, physical cues and integration. The general standards for data quality, reproducibility and deposition within the organoid community are also outlined. Lastly, we conclude by elaborating on the limitations of organoids in different applications, and the key priorities in organoid engineering for the coming years. Experimentation Cell sourceUnder defined physicochemical conditions, tissues such as small intestine 7 , colon 29,30 , stomach 31,32 , oesophagus 29 , tongue 33 , liver [34][35][36][37] , lung 14 , pancreas [38][39][40] , heart 41 , ear 42 and skin 43 have been obtained from iPSCs, adult or fetal cells and either stem/progenitor cells or differentiated cells. The starting cellular population for any given organoid is of prime importance, affecting not only the variability and heterogeneity in the structures obtained but also the function of the tissue they aim to model. To establish tissue-derived organoids or cancer organoids, tissue-resident stem/progenitor/differentiated cells or tumour cells, respectively, are obtained through an optimized tissue dissociation method. For iPSC-derived organoids, iPSC lines are established and fully characterized as the starting cells. Patient/tissue-derived stem cells are obtained through an optimized tissue dissociation method and then embedded into a 3D matrix mimicking stem cell niches. iPSCs can be maintained and expanded as undifferentiated clonal populations on feeder cells. To exemplify the generation of tissue-derived organoids we use intestinal organoids as an example (Fig. 2a), as this was the first tissue-derived organoid type established 7 . The small intestine and colon are opened longitudinally, washed and then cut into 2-4 mm fragments to increase the surface area for enzymatic digestion or further mechanical dissociation. EDTA treatment is used to chelate calcium, disrupting cell-cell adhesion and tissue integrity 44 . Larger tissue fragments and whole cells ...

Metastasis is clinically the most challenging and lethal aspect of breast cancer. While animal-based xenograft models are expensive and time-consuming, conventional two-dimensional (2D) cell culture systems fail to mimic in vivo signaling. In this study we have developed a three-dimensional (3D) scaffold system that better mimics the topography and mechanical properties of the breast tumor, thus recreating the tumor microenvironment in vitro to study breast cancer metastasis. Porous poly(ε-caprolactone) (PCL) scaffolds of modulus 7.0 ± 0.5 kPa, comparable to that of breast tumor tissue were fabricated, on which MDA-MB-231 cells proliferated forming tumoroids. A comparative gene expression analysis revealed that cells growing in the scaffolds expressed increased levels of genes implicated in the three major events of metastasis, viz., initiation, progression, and the site-specific colonization compared to cells grown in conventional 2D tissue culture polystyrene (TCPS) dishes. The cells cultured in scaffolds showed increased invasiveness and sphere formation efficiency in vitro and increased lung metastasis in vivo. A global gene expression analysis revealed a significant increase in the expression of genes involved in cell-cell and cell-matrix interactions and tissue remodeling, cancer inflammation, and the PI3K/Akt, Wnt, NF-kappaB, and HIF1 signaling pathways-all of which are implicated in metastasis. Thus, culturing breast cancer cells in 3D scaffolds that mimic the in vivo tumor-like microenvironment enhances their metastatic potential. This system could serve as a comprehensive in vitro model to investigate the manifold mechanisms of breast cancer metastasis.

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Inflammation in cancer fuels metastasis and worsens prognosis. Cancer-associated fibroblasts (CAFs) present in the tumor stroma play a vital role in mediating the cascade of cancer inflammation that drives metastasis by enhancing angiogenesis, tissue remodeling, and invasion. In vitro models that faithfully recapitulate CAF-mediated inflammation independent of coculturing with cancer cells are nonexistent. We have engineered fibrous matrices of poly(ε-caprolactone) (PCL) that can maintain the manifold tumor-promoting properties of patient-derived CAFs, which would otherwise require repetitive isolation and complex coculturing with cancer cells. On these fibrous matrices, CAFs proliferated and remodeled the extracellular matrix (ECM) in a parallel-patterned manner mimicking the ECM of high-grade breast tumors and induced stemness in breast cancer cells. The response of the fibroblasts was observed to be sensitive to the scaffold architecture and not the polymer composition. The CAFs cultured on fibrous matrices exhibited increased activation of the NF-κB pathway and downstream proinflammatory gene expression compared to CAFs cultured on conventional two-dimensional (2D) dishes and secreted higher levels of proinflammatory cytokines such as IL-6, GM-CSF, and MIP-3α. Consistent with this, we observed increased infiltration of inflammatory cells to the tumor site and enhanced invasiveness of the tumor in vivo when tumor cells were injected admixed with CAFs grown on fibrous matrices. These data suggest that CAFs better retain their tumor-promoting proinflammatory properties on fibrous polymeric matrices, which could serve as a unique model to investigate the mechanisms of stroma-induced inflammation in cancer progression.

The majority of the cancer-associated deaths is due to metastasis—the spread of tumors to other organs. Circulating tumor cells (CTCs), which are shed from the primary tumor into the circulation, serve as precursors of metastasis. CTCs have now gained much attention as a new prognostic and diagnostic marker, as well as a screening tool for patients with metastatic disease. However, very little is known about the biology of CTCs in cancer metastasis. An increased understanding of CTC biology, their heterogeneity, and interaction with other cells can help towards a better understanding of the metastatic process, as well as identify novel drug targets. Here we present a novel ex vivo 3D system for culturing CTCs from breast cancer patient blood samples using porous poly(ε-caprolactone) (PCL) scaffolds. As a proof of principle study, we show that ex vivo culture of 12/16 (75%) advanced stage breast cancer patient blood samples were enriched for CTCs identified as CK+ (cytokeratin positive) and CD45− (CD45 negative) cells. The deposition of extracellular matrix proteins on the PCL scaffolds permitted cellular attachment to these scaffolds. Detection of Ki-67 and bromodeoxyuridine (BrdU) positive cells revealed proliferating cell population in the 3D scaffolds. The CTCs cultured without prior enrichment exhibited dynamic differences in epithelial (E) and mesenchymal (M) composition. Thus, our 3D PCL scaffold system offers a physiologically relevant model to be used for studying CTC biology as well as for individualized testing of drug susceptibility. Further studies are warranted for longitudinal monitoring of epithelial–mesenchymal transition (EMT) in CTCs for clinical association.

Three-dimensional (3D) models have led to a paradigm shift in disease modeling in vitro, particularly for cancer. The past decade has seen a phenomenal increase in the development of 3D models for various types of cancers with a focus on studying stemness, invasive behavior, angiogenesis, and chemoresistance of cancer cells, as well as contributions of its stroma, which has expanded our understanding of these processes. Cancer biology is moving into exploring the emerging hallmarks of cancer, such as inflammation, immune evasion, and reprogramming of energy metabolism. Studies into these emerging concepts have provided novel targets and treatment options such as antitumor immunotherapy. However, 3D models that can investigate the emerging hallmarks are few and underexplored. As commonly used immunocompromised mice and syngenic mice cannot accurately mimic human immunology, stromal interactions, and metabolism and require the use of prohibitively expensive humanized mice, there is tremendous scope to develop authentic 3D tumor models in these areas. Taking the specific case of breast cancer, we discuss the currently available 3D models, their applications to mimic signaling in cancer, tumor−stroma interactions, drug responses, and assessment of drug delivery systems and therapies. We discuss the lacunae in the development of 3D tumor models for the emerging hallmarks of cancer, for lesser-explored forms of breast cancer, and provide insights to develop such models. We discuss how the next generation of 3D models can provide a better mimic of human cancer modeling compared to xenograft models and the scope toward preclinical models and precision medicine.

Tissue-engineering-based three-dimensional (3D) models offer several advantages over conventional two-dimensional (2D) cultures and can mimic tissues in vivo. Although studies have analyzed the changes in the expression of genes and proteins that might mediate in vivo-like signaling, the changes in the posttranscriptional control of gene expression that are critical in finetuning of signaling events has never been studied. In this study, we used next-generation sequencing (NGS) to analyze the changes in the post-transcriptional regulation in MDA-MB-231 breast cancer cells cultured on 3D scaffolds. The changes in the expression of several known microRNAs were similar to the changes reported in highly invasive cancers and their profiles highly correlated with xenotumors and human breast tumors. To elucidate the role of miRNAs in modulating metastatic potential, we integrated the miRNA and the mRNA microarray data and developed networks for major pathways implicated in metastasis. From these networks, we identified several key miRNA-mRNA interactions that might contribute to the invasive behavior and aid in developing a miRNA signature for highly invasive breast cancers. This report on the differential regulation of miRNAs in breast cancer cells cultured on scaffolds demonstrates that 3D culture better mimics the tissue in vivo with novel insights into the roles of miRNAs in modulating metastatic progression.

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