An improved method for culturing patient-derived colorectal cancer spheroids

Miyoshi, Hiroyuki; Maekawa, Hirofumi; Kakizaki, Fumihiko; Yamaura, Tadayoshi; Kawada, Kenji; Sakai, Yoshiharu; Taketo, Makoto Mark

doi:10.18632/oncotarget.25134

Cited by 34 publications

(54 citation statements)

References 38 publications

(49 reference statements)

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“…Our success rate in growing spheroids from primary tissue was at 88%, which is signi cantly higher than previously reported for work on patient-derived 3D models (e.g. Miyoshi et al reported a success rate of 73% in patient-derived colorectal spheroids (Miyoshi et al, 2018). Spheroids which failed to grow (4 out of 31) were mostly due to contamination with bacteria or fungi (in 3 cases), or low quality of the original tissue piece which resulted in a low yield of viable epithelial cells after single cell extraction (in 1 case).…”

Section: Discussioncontrasting

confidence: 59%

Patient-derived Tumor Spheroid Cultures as a Promising Tool to Assist Personalized Therapeutic Decisions

Hofmann

Cohen-Harazi

Maizels

et al. 2020

Preprint

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BackgroundBreast cancer is the most common cause of cancerrelated death in women. Treatment of breast cancer has many limitations including a lack of accurate biomarkers to predict success of chemotherapy, intrinsic resistance of a significant group of patients to the gold standard of therapy and the limited applicability of targeted therapy. Therefore, new tools are needed to provide doctors with guidance in choosing the most effective treatment plan for a particular patient and thus to increase the survival rate for breast cancer patients.MethodsHere, we present a successful method to grow in vitro spheroids from primary breast cancer tissue. Samples were received in accordance with relevant ethical guidelines and regulations. After tissue dissociation, in vitro spheroids were generated in a scaffold-free 96-well plate format. Spheroid composition was investigated by immunohistochemistry (IHC) of epithelial (Pan Cytokeratin (panCK)), stromal (Vimentin) and breast cancer-specific markers (ER, PR, Her2, GATA, Mammaglobin). Growth and cell viability of the spheroids was assessed upon treatment with multiple anti-cancer compounds. Student´s t-test and two-way ANOVA test were used for statistical analysis.ResultsWe were able to successfully grow spheroids from 27 out of 31samples from surgical resections of breast cancer tissuefrom previously untreated patients. Recapitulation of the histopathology of the tissue of origin was confirmed. Furthermore, a drug panel of standard first line chemotherapy drugs used to treat breast cancer was applied to assess the viability of the patient-derived spheroids and revealed variation in the response of the spheroids to different drug treatments. ConclusionsWe investigated the feasibility and the utility of an in vitro, patient-derived spheroid model for breast cancer therapy, and we conclude that spheroids serve as a highly efficient platform to explore cancer therapeutics and personalized treatment efficacy.These results have significant implications for the application of this model in clinical personalized medicine.

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Section: Discussioncontrasting

confidence: 59%

Patient-derived Tumor Spheroid Cultures as a Promising Tool to Assist Personalized Therapeutic Decisions

Hofmann

Cohen-Harazi

Maizels

et al. 2020

Preprint

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“…Recently, we established hCRC organoid/spheroid lines and patientderived tumor xenografts by an improved method (32,33). We examined 57 cases of hCRCs in which organoids or patient-derived tumor xenografts were successfully established.…”

Section: Il17rb Marks Mouse Intestinal Tscs In An Il-13-dependent Manmentioning

confidence: 99%

Lineage tracing and targeting of IL17RB ⁺ tuft cell-like human colorectal cancer stem cells

Goto

Fukuda

Yamaga

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Cancer stem cell (CSC)-specific markers may be potential therapeutic targets. We previously identified that Dclk1, a tuft cell marker, marks tumor stem cells (TSCs) in mouse intestinal adenomas. Based on the analysis of mouse Dclk1 + tumor cells, we aimed to identify a CSC-specific cell surface marker in human colorectal cancers (hCRCs) and validate the therapeutic effect of targeting it. IL17RB was distinctively expressed by Dclk1 + mouse intestinal tumor cells. Using Il17rb-CreERT2-IRES-EGFP mice, we show that IL17RB marked intestinal TSCs in an IL13-dependent manner. Tuft cell-like cancer cells were detected in a subset of hCRCs. In these hCRCs, lineage-tracing experiments in CRISPR-Cas9-mediated IL17RB-CreERT2 knockin organoids and xenograft tumors revealed that IL17RB marks CSCs that expand independently of IL-13. We observed up-regulation of POU2F3, a master regulator of tuft cell differentiation, and autonomous tuft cell-like cancer cell differentiation in the hCRCs. Furthermore, long-term ablation of IL17RBexpressing CSCs strongly suppressed the tumor growth in vivo. These findings reveal insights into a CSC-specific marker IL17RB in a subset of hCRCs, and preclinically validate IL17RB + CSCs as a cancer therapeutic target.C ancer stem cells (CSCs), capable of self-renewal and giving rise to progeny cells in cancers, have attracted attention as promising targets for anticancer therapeutics. The traditional xenotransplantation assays used to investigate the properties of CSCs have technical and conceptual limitations (1). Recent studies have shown that leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) marks CSCs in human colorectal cancers (hCRCs) in vivo by lineage tracing of LGR5-CreERT2 knockin hCRC organoids generated by CRISPR-Cas9-mediated gene editing (2, 3). Because most of the CSC markers are also expressed by normal stem cells (4-7), long-term CSC targeting may disrupt normal tissue homeostasis [e.g., liver injury after long-term LGR5-targeting (8)], whereas short-term CSC targeting leads to tumor regrowth (2,9). Hence, identification of CSC-specific markers is essential. We previously showed that Dclk1, a differentiated tuft cell marker in normal intestine, marks tumor stem cells (TSCs) in mouse intestinal adenomas by lineage-tracing experiments (10). However, whether DCLK1 marks CSCs in hCRCs has not been elucidated by in vivo lineage tracing of the tumors. Furthermore, feasible strategies to target DCLK1 + cells are necessary to realize a novel CSC-targeted therapy for hCRCs. Identification of a specific cell surface marker in Dclk1 + cells can facilitate the sorting analysis of Dclk1 + tumor cells and can also have the potential for future antibody therapeutics.The IL-17 receptor family includes five members (IL17RA to IL17RE) (11). The heterodimer of IL17RA and IL17RC serves as a receptor for IL-17A and IL-17F to mediate Th17 immune response, and the heterodimer of IL17RA and IL17RB serves as a receptor for IL-25 to mediate Th2 immune response (12).Recent studies showed that...

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“…Primary CRC specimens were obtained from patients who underwent colorectal resection and small pieces of tissue were transplanted into the subcutaneous tissues of immunodeficient female nude mouse (KSN/Slc mouse, SLC, Shizuoka, Japan) to establish PDXs. Spheroids were cultured as previously described [45]. These experiments were approved by the institutional Animal Ethics and Research Committee, Kyoto University (MedKyo 18148).…”

Section: Patient-derived Tumor Xenografts (Pdx) and Patient-derived Cmentioning

confidence: 99%

MicroRNA-9-5p-CDX2 Axis: A Useful Prognostic Biomarker for Patients with Stage II/III Colorectal Cancer

Nishiuchi

Hisamori

Sakaguchi

et al. 2019

Cancers

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A lack of caudal-type homeobox transcription factor 2 (CDX2) protein expression has been proposed as a prognostic biomarker for colorectal cancer (CRC). However, the relationship between CDX2 levels and the survival of patients with stage II/III CRC along with the relationship between microRNAs (miRs) and CDX2 expression are unclear. Tissue samples were collected from patients with stage II/III CRC surgically treated at Kyoto University Hospital. CDX2 expression was semi-quantitatively evaluated by immunohistochemistry (IHC). The prognostic impacts of CDX2 expression on overall survival (OS) and relapse-free survival (RFS) were evaluated by multivariable statistical analysis. The expression of miRs regulating CDX2 expression and their prognostic impacts were analyzed using The Cancer Genome Atlas Program for CRC (TCGA-CRC). Eleven of 174 CRC tissues lacked CDX2 expression. The five-year OS and RFS rates of patients with CDX2-negative CRC were significantly lower than those of CDX2-positive patients. Multivariate analysis of clinicopathological features revealed that CDX2-negative status is an independent marker of poor prognosis in stage II/III CRC. miR-9-5p was shown to regulate CDX2 expression. TCGA-CRC analysis showed that high miR-9-5p expression was significantly associated with poor patient prognosis in stage II/III CRC. In conclusion, CDX2, the post-transcriptional target of microRNA-9-5p, is a useful prognostic biomarker in patients with stage II/III CRC.

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An improved method for culturing patient-derived colorectal cancer spheroids

Cited by 34 publications

References 38 publications

Patient-derived Tumor Spheroid Cultures as a Promising Tool to Assist Personalized Therapeutic Decisions

Patient-derived Tumor Spheroid Cultures as a Promising Tool to Assist Personalized Therapeutic Decisions

Lineage tracing and targeting of IL17RB ⁺ tuft cell-like human colorectal cancer stem cells

MicroRNA-9-5p-CDX2 Axis: A Useful Prognostic Biomarker for Patients with Stage II/III Colorectal Cancer

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An improved method for culturing patient-derived colorectal cancer spheroids

Cited by 34 publications

References 38 publications

Patient-derived Tumor Spheroid Cultures as a Promising Tool to Assist Personalized Therapeutic Decisions

Patient-derived Tumor Spheroid Cultures as a Promising Tool to Assist Personalized Therapeutic Decisions

Lineage tracing and targeting of IL17RB + tuft cell-like human colorectal cancer stem cells

MicroRNA-9-5p-CDX2 Axis: A Useful Prognostic Biomarker for Patients with Stage II/III Colorectal Cancer

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Lineage tracing and targeting of IL17RB ⁺ tuft cell-like human colorectal cancer stem cells