Comparative Analysis of Patient-Matched PDOs Revealed a Reduction in OLFM4-Associated Clusters in Metastatic Lesions in Colorectal Cancer

Okamoto, Takuya; duVerle, David A.; Yaginuma, Katsuyuki; Natsume, Yasuko; Yamanaka, Hitomi; Kusama, Daisuke; Fukuda, Mayuko; Yamamoto, Mayuko; Perraudeau, Fanny; Srivastava, Upasna; Kashima, Yukie; Suzuki, Ayako; Kuze, Yuuta; Takahashi, Yu; Ueno, Masashi; Sakai, Yoshiharu; Noda, Tetsuo; Tsuda, Koji; Suzuki, Yutaka; Nagayama, Satoshi; Yao, Ryoji

doi:10.1016/j.stemcr.2021.02.012

Cited by 27 publications

(27 citation statements)

References 62 publications

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“…Cellular heterogeneity of metastatic CRC has been recently studied by bulk transcriptomic and scRNA-seq analyses of primary and metastatic PDOs. The authors found that metastatic PDOs show decreased expression of differentiated markers and, curiously, also of intestinal stem cell markers, demonstrating that primary and metastatic lesions have distinct cellular composition [ 93 ].…”

Section: Organoids As a Model To Study Crcmentioning

confidence: 99%

Organoids and Colorectal Cancer

Barbáchano

Fernández‐Barral

Bustamante-Madrid

et al. 2021

Cancers

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Organoids were first established as a three-dimensional cell culture system from mouse small intestine. Subsequent development has made organoids a key system to study many human physiological and pathological processes that affect a variety of tissues and organs. In particular, organoids are becoming very useful tools to dissect colorectal cancer (CRC) by allowing the circumvention of classical problems and limitations, such as the impossibility of long-term culture of normal intestinal epithelial cells and the lack of good animal models for CRC. In this review, we describe the features and current knowledge of intestinal organoids and how they are largely contributing to our better understanding of intestinal cell biology and CRC genetics. Moreover, recent data show that organoids are appropriate systems for antitumoral drug testing and for the personalized treatment of CRC patients.

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Section: Organoids As a Model To Study Crcmentioning

confidence: 99%

Organoids and Colorectal Cancer

Barbáchano

Fernández‐Barral

Bustamante-Madrid

et al. 2021

Cancers

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“…This protocol describes the knock-in of the marker cassette into the OLFM4 locus, which we identified as stem cell markers of PDOs established from CRC ( Okamoto et al., 2021 ). However, we have also successfully used this protocol to visualize and evaluate their progeny.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…PDOs were embedded into Matrigel on glass-bottom plates and cultured for three days with (B) or without AP20147 (C). Note that EGFP expression of PDOs was decreased (B), because OLFM4-postive stem cells reduced their self-replication potential late after passage ( Okamoto et al., 2021 ). …”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…The signal intensity of EGFP in PDOs mirrors the expression level of endogenous OLFM4 in the original clone ( Okamoto et al., 2021 ). OLFM4-positive cells were detected in a 3-dimensional image under confocal laser scanning microscopy ( Figure 5 A).…”

Section: Expected Outcomesmentioning

confidence: 98%

“…For complete details on the generation and use of this protocol, please refer to Okamoto et al. Okamoto et al. (2021) .…”

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A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal cancer patient-derived organoids

et al. 2021

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Summary Patient-derived organoids (PDOs) recapitulate the cellular heterogeneity of the original colorectal tumor tissue. Here, we describe a protocol to generate genetically modified PDOs to investigate cancer stem cells. This protocol uses the CRISPR-Cas9 system to knock-in the IRES-EGFP-P2A-iCaspase9 cassette into the 3′ UTR of the potential cancer stem cell marker gene, which allows us to investigate their potential for self-replication and pluripotency. We describe the procedure for generating mutant PDOs and their application for stem cell research. For complete details on the generation and use of this protocol, please refer to Okamoto et al. Okamoto et al. (2021) .

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Olfactomedin 4 associates with expression of differentiation markers but not with properties of cancer stemness, EMT nor metastatic spread in colorectal cancer

Jaitner

Pretzsch

Neumann

et al. 2022

The Journal of Pathology CR

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Tumor stem cells play a pivotal role in carcinogenesis and metastatic spread in colorectal cancer (CRC). Olfactomedin 4 (OLFM4) is co-expressed with the established stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 at the bottom of intestinal crypts and has been suggested as a surrogate for cancer stemness and a biomarker in gastrointestinal tumors associated with prognosis. Therefore, it was the aim of the present study to clarify whether OLFM4 is involved in carcinogenesis and metastatic spread in CRC. We used a combined approach of functional assays using forced OLFM4 overexpression in human CRC cell lines, xenograft mice, and an immunohistochemical approach using patient tissues to investigate the impact of OLFM4 on stemness, canonical Wnt signaling, properties of metastasis and differentiation as well as prognosis. OLFM4 expression correlated weakly with tumor grade in one patient cohort (metastasis collection: p = 0.05; pooled analysis of metastasis collection and survival collection: p = 0.19) and paralleled the expression of differentiation markers (FABP2, MUC2, and CK20) (p = 0.002) but did not correlate with stemness-associated markers. Further analyses in CRC cells lines as well as xenograft mice including forced overexpression of OLFM4 revealed that OLFM4 neither altered the expression of markers of stemness nor epithelialmesenchymal transition, nor did OLFM4 itself drive proliferation, migration, or colony formation, which are all prerequisites of carcinogenesis and tumor progression. In line with this, we found no significant correlation between OLFM4 expression, metastasis, and patient survival. In summary, expression of OLFM4 in human CRC seems to be characteristic of differentiation marker expression in CRC but is not a driver of carcinogenesis nor metastatic spread.

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Comparative Analysis of Patient-Matched PDOs Revealed a Reduction in OLFM4-Associated Clusters in Metastatic Lesions in Colorectal Cancer

Cited by 27 publications

References 62 publications

Organoids and Colorectal Cancer

Organoids and Colorectal Cancer

A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal cancer patient-derived organoids

Olfactomedin 4 associates with expression of differentiation markers but not with properties of cancer stemness, EMT nor metastatic spread in colorectal cancer

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