Using 3D Organoid Cultures to Model Intestinal Physiology and Colorectal Cancer

Short, Sarah P.; Costacurta, Patricia; Williams, Christopher S.

doi:10.1007/s11888-017-0363-8

Cited by 12 publications

(12 citation statements)

References 86 publications

(103 reference statements)

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“…Additionally, in order to expedite the discovery of orally available drugs, methods to shorten the time to reach a fully differentiated confluent Caco-2 cell monolayer are highly desirable 23 . As has been previously shown, moving cells from a 2D to a 3D environment and restoring interactions with the ECM can induce dramatic effects on gene expression, differentiation, and metabolism and can, thus, more accurately model the in vivo phenotype 24 .…”

Section: Discussionmentioning

confidence: 96%

Human intestinal spheroids cultured using Sacrificial Micromolding as a model system for studying drug transport

Samy

Levy

Phong

et al. 2019

Sci Rep

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In vitro models of the small intestine are crucial tools for the prediction of drug absorption. The Caco-2 monolayer transwell model has been widely employed to assess drug absorption across the intestine. However, it is now well-established that 3D in vitro models capture tissue-specific architecture and interactions with the extracellular matrix and therefore better recapitulate the complex in vivo environment. However, these models need to be characterized for barrier properties and changes in gene expression and transporter function. Here, we report that geometrically controlled self-assembling multicellular intestinal Caco-2 spheroids cultured using Sacrificial Micromolding display reproducible intestinal features and functions that are more representative of the in vivo small intestine than the widely used 2D transwell model. We show that Caco-2 cell maturation and differentiation into the intestinal epithelial phenotype occur faster in spheroids and that they are viable for a longer period of time. Finally, we were able to invert the polarity of the spheroids by culturing them around Matrigel beads allowing superficial access to the apical membrane and making the model more physiological. This robust and reproducible in vitro intestinal model could serve as a valuable system to expedite drug screening as well as to study intestinal transporter function.

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Section: Discussionmentioning

confidence: 96%

Human intestinal spheroids cultured using Sacrificial Micromolding as a model system for studying drug transport

Samy

Levy

Phong

et al. 2019

Sci Rep

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“…Accordingly HT-29 spheroids with compact round shape may be more suitable platforms for anti-cancer drug testing in CRC than Caco-2 spheres. In general, in order to create more practical and functional CRC spheroid models, it is essential to characterize the gene expression alterations, invasion, and drug transporters in spheroids to standardize them based on research requirements [61,64,[71][72][73]. Hence, CRC spheroid models were further assessed to determine their potential in enrichment of CSCs-related characteristics, including the cell surface marker patterns, serial sphere formation capability, and gene expression pro les of multipotency, EMT and drug resistance transporters compared to the parental cells.…”

Section: Discussionmentioning

confidence: 99%

Morphological and Molecular Characteristics of Spheroid Formation in HT-29 and Caco-2 Colorectal Cancer Cell Lines

Gheytanchi

Naseri

Karimi‐Busheri

et al. 2021

Preprint

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Background: Relapse and metastasis in colorectal cancer (CRC) are often attributed to cancer stem-like cells (CSCs), as small sub-population of tumor cells with ability of drug resistance. Accordingly, development of appropriate models to investigate CSCs biology and establishment of effective therapeutic strategies is warranted. Hence, we aimed to assess the capability of two widely used and important colorectal cancer cell lines, HT-29 and Caco-2, in generating spheroids and their detail morphological and molecular characteristics.Methods: CRC spheroids were developed using hanging drop and forced ﬂoating in serum-free and non-attachment condition and their morphological features were evaluated by scanning electron microscopy (SEM). Then, the potential of CSCs enrichment in spheroids was compared to their adherent counterparts by analysis of serial sphere formation capacity, real-time PCR of key stemness genes (KLF4, OCT4, SOX2, NANOG, C-MYC) and the expression of potential CRC-CSCs surface markers (CD166, CD44, and CD133) by flow cytometry. Finally, the expression level of some EMT-related (Vimentin, SNAIL1, TWIST1, N-Cadherin, E-Cadherin, ZEB1) and multi-drug resistant (ABCB1, ABCC1, ABCG2) genes was evaluated.Results: Although with different morphological features, both cell lines were formed CSCs-enriched spheroids, indicated by ability to serial sphere formation, significant up-regulation of stemness genes, SOX2, C-MYC, NANOG and OCT4 in HT-29 and SOX2, C-MYC and KLF4 in Caco-2 spheroids (p-value<0.05) and increased expression of CRC-CSC markers compared to parental cells (p-value<0.05). Additionally, HT-29 spheroids exhibited a signiﬁcant higher expression of both ABCB1 and ABCG2 (p-value=0.02). The significant up-regulation of promoting EMT genes, ZEB1, Twist1, E-cadherin and SNAIL1 in HT-29 spheroids (p-value=0.03), SNAIL1 and Vimentin in Caco-2 spheroids (p-value<0.05) and N-cadherin down-regulation in both spheroids were observed.Conclusion: Based on enrichment of CSC-related features in HT-29 and Caco-2 spheroids, our findings suggest CRC spheroids culture as simple, cost-effective and efficient model to imitate the complexity of in vivo CRC tumors including self-renewal, drug resistance and invasion potential for CSC research.

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“…To determine the pathophysiologic translation of these observations to CRC, we next utilized a 3D culture system of primary human CRC samples. The 3D tumor organoid approach has been increasingly used in recent years to model tumor cell behavior and therapeutic response, as organoids have been shown to more closely maintain morphology, histology, and cell heterogeneity than 2D cell lines (21,37). Human tumor organoids were established from primary CRC tumors (Supplemental Table 2), and 3 independent lines expressing detectable levels of STK17A (referred to as HTO1, HTO2, and HTO3) were selected for STK17A KD (Supplemental Figs.…”

Section: Resultsmentioning

confidence: 99%

Serine Threonine Kinase 17A Maintains the Epithelial State in Colorectal Cancer Cells

Short

Thompson

Bilotta

et al. 2019

Molecular Cancer Research

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Serine Threonine Kinase 17A (STK17A) is a ubiquitously expressed kinase originally identified as a regulator of apoptosis; however, whether it functionally contributes to colorectal cancer (CRC) has not been established. Here, we have analyzed STK17A in CRC and demonstrate decreased expression of STK17A in primary tumors which is further reduced in metastatic lesions, indicating a potential role in regulating the metastatic cascade. Interestingly, changes in STK17A expression did not modify proliferation, apoptosis, or sensitivity of CRC cell lines to treatment with the chemotherapeutic 5-fluorouracil. Instead, STK17A knockdown induced a robust mesenchymal phenotype consistent with the epithelial-mesenchymal transition, including spindle-like cell morphology, decreased expression of adherens junction proteins, and increased migration and invasion. Additionally, overexpression of STK17A decreased cell size and induced wide-spread membrane blebbing, a phenotype often associated with activation of cell contractility. Indeed, STK17A overexpressing cells displayed heighted phosphorylation of myosin light chain in a manner dependent on STK17A catalytic activity. Finally, patient-derived tumor organoid cultures were used to more accurately determine STK17A’s effect in primary human tumor cells. Loss of STK17A induced morphological changes, decreased E-cadherin, increased invasion, and augmented organoid attachment on 2D substrates, all-together suggesting a more metastatic phenotype. Collectively, these data indicate a novel role for STK17A in regulation of epithelial phenotypes and indicate its functional contribution to CRC invasion and metastasis. Implications: Loss of serine threonine kinase 17A occurs in colorectal cancer metastasis, induces mesenchymal morphologies, and contributes to tumor cell invasion and migration in colorectal cancer.

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Using 3D Organoid Cultures to Model Intestinal Physiology and Colorectal Cancer

Cited by 12 publications

References 86 publications

Human intestinal spheroids cultured using Sacrificial Micromolding as a model system for studying drug transport

Human intestinal spheroids cultured using Sacrificial Micromolding as a model system for studying drug transport

Morphological and Molecular Characteristics of Spheroid Formation in HT-29 and Caco-2 Colorectal Cancer Cell Lines

Serine Threonine Kinase 17A Maintains the Epithelial State in Colorectal Cancer Cells

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