Sequential cancer mutations in cultured human intestinal stem cells.
Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.
1] Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, The Netherlands [2] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands.
Jarno
J
Drost
1] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands [2] Molecular Cancer Research, Centre for Molecular Medicine, UMC Utrecht, 3584CG, Utrecht, The Netherlands.
Richard H
RH
van Jaarsveld
1] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands [2] Molecular Cancer Research, Centre for Molecular Medicine, UMC Utrecht, 3584CG, Utrecht, The Netherlands.
Bas
B
Ponsioen
1] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands [2] Laboratory of Experimental Oncology and Radiobiology, Centre for Experimental Molecular Medicine, AMC, 1105AZ Amsterdam, The Netherlands.
Cheryl
C
Zimberlin
1] Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, The Netherlands [2] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands.
Ruben
R
van Boxtel
Department of Medical Genetics, UMC Utrecht, 3508AB Utrecht, The Netherlands.
Arjan
A
Buijs
1] Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, The Netherlands [2] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands.
Norman
N
Sachs
1] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands [2] Molecular Cancer Research, Centre for Molecular Medicine, UMC Utrecht, 3584CG, Utrecht, The Netherlands.
René M
RM
Overmeer
Department of Pathology, UMC Utrecht, 3584CX Utrecht, The Netherlands.
G Johan
GJ
Offerhaus
1] Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, The Netherlands [2] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands.
Harry
H
Begthel
1] Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, The Netherlands [2] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands.
Jeroen
J
Korving
1] Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, The Netherlands [2] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands [3] Foundation Hubrecht Organoid Technology (HUB), 3584CT Utrecht, The Netherlands.
Marc
M
van de Wetering
1] Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, The Netherlands [2] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands.
Gerald
G
Schwank
1] Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, The Netherlands [2] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands.
Meike
M
Logtenberg
1] Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, The Netherlands [2] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands.
Edwin
E
Cuppen
1] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands [2] Molecular Cancer Research, Centre for Molecular Medicine, UMC Utrecht, 3584CG, Utrecht, The Netherlands.
Hugo J
HJ
Snippert
1] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands [2] Laboratory of Experimental Oncology and Radiobiology, Centre for Experimental Molecular Medicine, AMC, 1105AZ Amsterdam, The Netherlands.
Jan Paul
JP
Medema
1] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands [2] Molecular Cancer Research, Centre for Molecular Medicine, UMC Utrecht, 3584CG, Utrecht, The Netherlands.
Geert J P L
GJ
Kops
1] Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, The Netherlands [2] Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, The Netherlands.
Hans
H
Clevers
D000782:Aneuploidy; D000818:Animals; D064113:CRISPR-Cas Systems; D002648:Child; D002675:Child, Preschool; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D005260:Female; D017491:Genes, APC; D016158:Genes, p53 / Q000235:genetics; D064593:Heterografts; D006801:Humans; D007093:Imidazoles; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000473:pathology*; D051379:Mice; D008875:Middle Aged; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D009361:Neoplasm Invasiveness / Q000235:genetics / Q000473:pathology; D009368:Neoplasm Transplantation; D009940:Organoids / Q000378:metabolism* / Q000473:pathology*; D010879:Piperazines; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism; D051901:Smad4 Protein / Q000172:deficiency; D055153:Stem Cell Niche / Q000502:physiology; D013234:Stem Cells / Q000378:metabolism / Q000473:pathology*
D007093:Imidazoles; D036341:Intercellular Signaling Peptides and Proteins; C117307:KRAS protein, human; D010879:Piperazines; C497796:SMAD4 protein, human; D051901:Smad4 Protein; C482205:nutlin 3; D016283:Proto-Oncogene Proteins p21(ras)