Quick Review On Colon Organoids And Colon Epithelial Organoid Culture Recipe

by Dolly the Sheep | Mar 9, 2022 | Cancer, Organoids, Stem Cell

The Colon Organoids

Using organoids as a model to study colorectal cancer
Organoid limitations
Organoids to study the intestinal microbiome and host-pathogen interactions
Perspectives
Selected compositions for colon epithelial organoid culture recipe by Lab-A-Porter

Colorectal cancer is one of the most frequent and lethal types of cancer. Organoids were recently established as a new system to culture normal and tumour cells collected from patients subjected to surgery or endoscopic tests. Organoids are being used to analyze the molecular and genetic bases of colorectal cancer initiation and progression. In the recent review article published by Cancers, Barbachano al et described the use of patient-derived organoids to examine the tumorigenic process, including immunotherapy, interplay with microorganisms and assays of drug treatments at an individualized level. The authors had also summarized the most recent developments of what is known as organoid technology directed towards personalized medicine.

Using organoids as a model to study colorectal cancer

1. Tools for modelling and cell plasticity analyses Organoid systems have become a suitable model to examine in vivo tumour evolution and cancer stem cell plasticity by combining gene editing and xenotransplantation in mice. 2. Tools to research molecular and genetic aspects Human and mouse colon organoids have replaced colon carcinoma cell lines as a system to study the role of genes, pathways, compounds, and agents in colorectal cancer. Organoids have been heavily used to study the Wnt/beta-catenin pathway:

Using mouse organoids from three intestinal regions, Wnt signalling has been found to have a variable effect on gene expression and downregulate differentiation genes in a region-specific fashion.
Using human intestinal patient-derived organoids and patient-derived tumour organoids which were genetically modified by means of CRISPR-Cas9, have been used to examine the role of genes and factors in colorectal cancer progression.
- - The authors have used organoid technology to determine possible differences between colon and rectum tumours.

3. Platform for screening assays 3.1 Tools for toxicity studies

Failures in many clinical trials are mostly related to problems with efficacy or harmful side effects.
Lu and colleagues successfully used mouse small intestine organoid cultures to study drug metabolism and toxicity.

3.2 Tools for drug research and discovery

Ashley and colleagues were the first to perform a proof-of-concept study using primary cultures of colorectal cancer cells for preclinical drug testing.
Other groups used colorectal cancer organoid biobanks to perform high-throughput analyses to identify potential inhibitors oncogenes and to study the regenerative response of normal intestinal organoids to nearly 3000 compounds.
Recently, Huelsken’s group has established a high-content high-throughput screening system in small intestine mouse organoids to identify small-molecule drugs that are able to induce the differentiation of intestinal wild-type and cancer cells.

3.3 Tools to research personalized treatment

Organoids are the only system that allows the long-term culture of patient stem cells in vitro, making it possible to replicate ex vivo primary cancers, infectious or developmental diseases directly from a biopsy of the patient.
- - Clever’s group generated rectal organoids directly from cystic fibrosis patients and analyzed their functional response to available therapeutic agents in the intro
Organoids can be used as tools to predict clinical outcomes in colorectal cancer.
- - Colonic organoids derived from human iPSCs have been used for colorectal cancer drug testing.
  - Potential to reduce inefficient medication, deleterious side-effects, and health care costs.

Organoid limitations

The success rate for patient-derived tumour organoids is impacted by the characteristics of the tumour and the difficulty to predict an adequate culture medium for each tumour.

Intratumor heterogeneity is another issue to consider.
- - The maintenance of substantial genetic stability and genetic heterogeneity over the course of the culture period cannot be predicted.

Tumours are dynamic entities that change over time in therapy-naïve patients.
- - Organoids established at a one-time point only faithfully represent the tumour at the precise developmental time of their establishment.

Issues about matrix selection
- Animal origin of extracellular matrix
- Adequacy of cell binding to matrix
- Whether specific composition resembles the niche conditions and the induction of physiological signalling
- Cytotoxicity
- Optimal stiffness
- Batch variability

The structure of adipose-derived stem cell patient-derived organoids is made exclusively of epithelial cells and lack the stroma: fibroblasts, immune cells, nerves, and vessels.

Organoids to study the intestinal microbiome and host-pathogen interactions

- Patient-derived organoids have been described as a model to study host-pathogen interaction, including the role of the microbiome in colorectal cancer.
  - - A recent study by Clever’s group has demonstrated a direct role of the genotoxic pks+strain of E-coli in colorectal cancer tumorigenesis.
- Intestinal organoids are emerging as a model system to investigate host-parasite and host-virus interactions.
- Infection by several enteroviruses and coronaviruses have been investigated including SARS-CoV-2.

Perspectives

- Despite several drawbacks and limitations, patient-derived organoids are today considered the preferential model to study colorectal cancer.
- In combination with cutting-edge techniques such as CRISPR, single-cell RNA sequencing and orthotopic xenotransplantation, organoids may significantly contribute to colorectal cancer modelling and knowledge.
- Improvements in the field should come from the optimization of organoids with the incorporation of stromal components and the generation of organoid-on-a-chip devices.
- Available data and ongoing studies point towards the desired precision treatments that will hopefully contribute to improving the outcome of colorectal cancer patients in the future.

Selected compositions for colon epithelial organoid culture recipe by Lab-A-Porter

EGF

EGF is a potent growth factor that stimulates the proliferation of various epidermal and epithelial cells. It has been shown to inhibit gastric secretion and be involved in wound healing.

Noggin

Noggin belongs to a group of diffusible proteins that bind to ligands of the TGF-β family and regulate their activity by inhibiting their access to signalling receptors.

R-Spondin-1

R-Spondin-1 is expressed in certain areas of the developing central nervous system, as well as in the adrenal glands, ovary, testis, thyroid, and trachea.

Wnt-3a

Wnt-3a signalling is essential for various morphogenetic events, including embryonic patterning, cell determination, cell proliferation, CNS development, and cytoskeletal formation.

Y 27632

Y-27632 is a selective inhibitor of Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) family of protein kinases that selectively competes with ATP for binding to the catalytic site.

A 83-01

A 83-01 is a potent activating receptor-like kinase inhibitor (ALK4 IC50 45nM, ALK5 IC50 12nM, ALK7 IC50 7.5nM). It is a more potent inhibitor of ALK5 than SB 431542 and is reported to inhibit Smad signalling and epithelial-to-mesenchymal transition by transforming growth factor-beta.

Nicotinamide

Nicotinamide is an amide of nicotinic acid, a vitamin of the B complex. In cells, it is incorporated into NADP+ and NAD+, coenzymes for a wide variety of enzymatic oxidation-reduction reactions.

SB 202190

SB 202190 is a selective and potent p38 MAP kinases inhibitor, binding to its ATP pocket. It has negligible inhibiting properties on other MAP kinases such as ERKs and JNKs.

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References:

Barbáchano, Antonio et al. “Organoids and Colorectal Cancer.” Cancers vol. 13,11 2657. 28 May. 2021, doi:10.3390/cancers13112657

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