新型传统锯齿状腺瘤类器官模型
创作:Lexi 审核:Lexi 2019年10月28日
  • 实施类器官染色体工程需要破坏TP53基因,IGF1和FGF2可促使人正常结肠类器官产生R-脊椎蛋白基因融合;
  • 表达BRAFV600E和R-脊椎蛋白融合基因的类器官可形成平的锯齿状病灶;
  • R-脊椎蛋白基因融合的结肠类器官在组织学上类似传统的锯齿状腺瘤(TSAs)前体;
  • GREM1在TSAs中被激活,并增加结肠类器官中LGR5的表达,异种移植过表达GREM1的R-脊椎蛋白融合基因类器官可模拟TSA;
  • 包括LGR5阴性细胞在内的多种细胞群体均有致癌潜力。
主编推荐语
Lexi
传统的锯齿状腺瘤(TSAs)是一种罕见的结直肠息肉,具有独特的组织学特征。虽然在TSAs中发现了R-脊椎蛋白基因融合,但由于缺乏人体组织的染色体工程平台,目前尚不清楚这些现象是否足以发展为TSA。最新发表在Gastroenterology杂志的文章利用CRISPR-Cas9介导的染色体和人结肠类器官的遗传修饰,研究了在TSA中发现的R-脊椎蛋白基因融合和其他遗传改变的影响。开发出有效的染色体工程改造的人正常结肠器官,该模型可用于RSPO融合基因和GREM1过表达的人结直肠癌的进展研究。
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Gastroenterology [IF:17.373]

Chromosome Engineering of Human Colon-Derived Organoids to Develop a Model of Traditional Serrated Adenoma

利用人结肠来源类器官的染色体工程开发传统锯齿状腺瘤模型

10.1053/j.gastro.2019.10.009

2019-10-14, Article

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Background & Aims: Traditional serrated adenomas (TSAs) are rare colorectal polyps with unique histologic features. Fusions in R-spondin genes have been found in TSAs, but it is not clear whether these are sufficient for TSA development, due to the lack of a chromosome engineering platform for human tissues. We studied the effects of fusions in R-spondin genes and other genetic alterations found in TSA using CRISPR-Cas9–mediated chromosome and genetic modification of human colonic organoids.
Methods: We introduced chromosome rearrangements that involve R-spondin genes into human colonic organoids, with or without disruption of TP53, using CRISPR-Cas9 (chromosome-engineered organoids). We then knocked a mutation into BRAF encoding the V600E substitution and overexpressed the GREM1 transgene; the organoids were transplanted into colons of NOG mice and growth of xenograft tumors was measured. Colon tissues were collected and analyzed by immunohistochemistry or in situ hybridization. We also established 2 patient-derived TSA organoid lines and characterized their genetic features and phenotypes. We inserted a bicistronic cassette expressing a dimerizer-inducible suicide gene and fluorescent marker downstream of the LGR5 gene in the chromosome-engineered organoids; addition of the dimerizer eradicates LGR5+ cells. Some tumor-bearing mice were given intraperitoneal injections of the dimerizer to remove LGR5-expressing cells.
Results: Chromosome engineering of organoids required disruption of TP53 or culture in medium containing IGF1 and FGF2. In colons of mice, organoids that expressed BRAFV600E and fusions in R-spondin genes formed flat serrated lesions. Patient-derived TSA organoids grew independent of exogenous R-spondin, and 1 line grew independent of Noggin. Organoids that overexpressed GREM1, in addition to BRAFV600E and fusions in R-spondin genes, formed polypoid tumors in mice that had similar histologic features to TSAs. Xenograft tumors persisted after loss of LGR5-expressing cells.
Conclusions: We demonstrated efficient chromosomal engineering of human normal colon organoids. We introduced genetic and chromosome alterations into human colon organoids found in human TSAs; tumors grown from these organoids in mice had histopathology features of TSAs. This model might be used to study progression of human colorectal tumors with RSPO fusion gene and GREM1 overexpression.

First Authors:
Kenta Kawasaki

Correspondence Authors:
Toshiro Sato

All Authors:
Kenta Kawasaki,Masayuki Fujii,Shinya Sugimoto,Keiko Ishikawa,Mami Matano,Yuki Ohta,Kohta Toshimitsu,Sirirat Takahashi,Naoki Hosoe,Shigeki Sekine,Takanori Kanai,Toshiro Sato

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