Organoid-based high-throughput RNA sequencing aids search for colorectal cancer drugs

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 Organoid-based high-throughput RNA sequencing aids search for colorectal cancer drugs

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 Organoid-based high-throughput RNA sequencing aids search for colorectal cancer drugs. 

Colorectal cancer (CRC) is one of the three most frequently diagnosed cancers in the world today and one of the leading causes of cancer-related morbidity and mortality.

Despite the continuous development of cancer therapy in recent years, there is still a lack of effective treatments for patients with advanced and metastatic bowel cancer, thus new drug-based therapies are urgently needed.

source: www.cell.com

In a recent study, the team of Joerg Huelsken from the Swiss Bowel Cancer Institute developed an organoid-based high-throughput screening system “TORNADO-seq” for the identification of small molecules that can induce the differentiation of normal and cancer cells in the gut drug.

Specifically, the method achieves research goals by quantifying RNA expression analysis of a large number of cell-type-specific genes, followed by deconvolution methods to infer cell-type composition.

As a high-content technique, mRNA expression analysis offers many advantages over other methods, such as antibody- or reporter gene-based methods, such as precision, scalability, and sensitivity.

This method can not only discover drugs, but also further reveal the underlying biological mechanisms.

The high-content assay developed by the authors provides information on the frequency of all possible gut cell phenotypes, major signaling pathways, and general cell physiology by measuring gene expression profiles in organoids derived from healthy gut or cancerous gut Expression levels of 206 genes.

Figure 1. Schematic diagram of intestinal organoid differentiation and establishment of RNA-seq analysis process

To cover different intestinal cell phenotypes, the authors selected marker genes from 8 populations: ISC, transiently expanding (TA) cells, absorptive intestinal epithelial cells (E), EEC, goblet cells, Paneth cells (P) , cluster cells and static ISCs (qISCs).

These marker genes were picked from publicly available databases (RNA-seq of gastrula cell suspensions and organoid cultures and single-cell RNA-seq).

Through further enrichment, fluorescence-activated cell sorting (FACS) or in vitro differentiation protocols, the authors additionally included 58 genes reporting major signaling pathways (Notch, Wnt, Hedgehog, Hippo, BMP/TGF-b], NF-kB, etc. ), 27 general cell physiology genes (including cell cycle, metabolism, angiogenesis, apoptosis, epithelial-mesenchymal transition, etc.), and 6 housekeeping genes.

Further, the authors screened for drug molecules that can promote cell differentiation in healthy intestinal organoids. The results showed that among the drugs with the highest hit rate were:

1. Molecules already used to treat colon cancer – itraconazole, pyrrolidine,

2. Tubulin inhibitors (bendazole, colchicine) ),

3. cytotoxic drugs known to affect general cell physiology (antimetabolites gemcitabine, azaguanine, mercaptopurine, floxuridine topoisomerase inhibitors,

4. cytotoxic antibiotics (anthracyclines and Antimycin).

In addition, the screening program identified several drug candidates such as the antipsychotic phenothiazine, cholesterol-lowering statins, antifungal conazole, selective estrogen receptor modulators (SERMs), Glucocorticoids and antihistamines, etc.

Figure 2. Effects of different drugs on the expression levels of genes related to cell differentiation in healthy intestinal organoids

The authors then compared the effects of different drugs on cell differentiation in healthy intestinal organoids versus cancerous ones.

The results showed that 16 of the 27 drugs that caused the differentiation of healthy intestinal cells also caused the differentiation of cancerous intestinal cells (the expression levels of stem cell-related markers were decreased, accompanied by an increase in differentiation-related markers), while a large number Drug molecules that are ineffective against the differentiation of healthy intestinal cells are also ineffective against the differentiation of cancerous intestinal cells.

This result suggests a necessary link between a drug’s effectiveness and its ability to affect cell differentiation.

In conclusion, the authors developed targeted organoid sequencing (TORNADO-seq) technology to monitor the expression of mainstream “gene markers” in high-throughput for detailed assessment of cellular phenotypes in organoids.

TORNADO-seq serves as a fast, robust, and inexpensive ($5 per sample) method to examine mixtures of cells and their differentiation status in the intestinal system. Using this approach, the authors identified drugs that promote the phenotype of differentiated cells and showed that these drugs have high efficacy against cancer.

In addition, TORNADO-seq provides insight into how these drugs work. This technique can be easily adapted to many other systems and allows a more systematic, large-scale and quantitative approach to study the biology of complex cellular systems.

Reference:

Maxim Norkin, Paloma Ordóñez-Morán, Joerg Huelsken et al., High-content, targeted RNA-seq screening in organoids for drug discovery in colorectal cancer. Cell Reports (2021)
DOI: https://doi.org/10.1016/j .celrep.2021.109026

 Organoid-based high-throughput RNA sequencing aids search for colorectal cancer drugs

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