Cancer Stem Cells from Colorectal Cancer Cell Lines. Walter Bodmer Weatherall Institute of Molecular Medicine Oxford University
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1 Cancer Stem Cells from Colorectal Cancer Cell Lines Walter Bodmer Weatherall Institute of Molecular Medicine Oxford University
2 Fully differentiated cells: columnar goblet enteroendocrine Myofibroblasts transit amplifying or progenitor cells stem cells
3 Cetuximab Response Resistant Partially Resistant Sensitive SYMBOL KRAS BRAF PIK3CA PTEN HDC135 N N E542K N HRA19 N N E542K N VACO429 N N N N CACO2 N N N N COLO206 N N N LIM1863 N N N N RKO N V600E H1047R N HCT116 G13D N H1047R N CCO7 G13D N N N COLO201 N V600E N N COLO320 DM N N N N HDC8 G12D N N N SW480 G12V N N N SW620 G12V N N N RCM1 G12V N N N OUMS23 CC20 N N N N LS180 G12D N H1047R N NCIH716 N N N N LS411 N V600E N N HT29 N V600E N N OXCO1 N V600E N N COLO741 N V600E N N VACO4S G12V N N N HCT15 G13D N E545K N C125PM N N N N LS1034 N N N N C10 N N N N SW948 N N E542K N T84 G13D N E542K N SKCO1 G12V N N N HCC2998 N N N N VACO5 N V600E H1047R N SW1116 G12A N N N LS123 G12S N N N C80 N N N N VACO10M S N N N N NCIH747 G13D N N N LOVO G13D N N N DLD1 * N E542K N SW1417 N V600E N N PMFKO14 N N N N SW837 G12C N N N SW1222 N N N LS513 G12D N N N LS174T G12D N H1047R N VACO400 N/A N/A N/A N HT55 N N N N HDC111 G12S N E545K N VACO4A G12V N N N SYMBOL KRAS BRAF PIK3CA PTEN C32 N N N N PCJW N/A N/A N/A N SW403 G12V N N N HDC9 N N N N CAR1 N N N N OXCO3 N N N N GP2D G12D N H1047L N OXCO2 N N N N HCA7 N N N N C106 N N N N COLO678 G12D N N N C84 G12A N N N SYMBOL KRAS BRAF PIK3CA PTEN SW48 N N N N HDC114 N N N N HCA46 N N N N HDC54 N N N N HDC57 N N N N CCK81 N N N N HDC73 N N N N HDC142 N N N N HDC82 N N N N C99 N N N N NCIH508 N N E545K N
4 Lines are good representatives of primary tumours Spectrum of mutations in cancers same as in lines eg p53 72%, APC 80%, -catenin 12%, KRAS 40%, BRAF 13% Same mutations in line and original primary tumour Lines and tumours show similar patterns of chromosomal gains and losses by acgh Patterns of methylation and mrna expression in lines and tumours very similar Lines do not change in culture: Duplicate lines have same mutations even after many separate culture generations Whole genome expression data clusters duplicates most closely together Duplicates mostly show very similar methylation and drug response profiles, even though duplicates from same tumour may be chosen to be different attached or not, primary versus metastasis Lines show similar drug responses to what is seen in patients Lines enable functional studies, are not contaminated with normal tissue
5 Single Cells from the SW1222 Cell Line Form Colonies with Different Morphologies when Grown in 3D. Yeung et al, PNAS, 2010 SW1222 Megacolony Richman and Bodmer1988 SW1222 Small Colony
6 Cells from SW1222 Megacolonies are more Clonogenic and can Regenerate Megacolonies and Small Colonies NOD/SCID mice 200 cells from Megacolonies 1000 cells from Small colonies 1 Tumour No tumour 2 Tumour No tumour 3 Tumour No tumour 4 No tumour No tumour Total 3/4 0/4 Cells From Small Colonies Only Give Rise to More Small Colonies. Cells from megacolonies are able to initiate tumors in NOD/SCID mice, but cells from small colonies are not tumorigenic Yeung et al, PNAS, 2010
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8 AUA-1 EpCAM CDX-1 Chromogranin PR4D1 Mucin
9 Colorectal Cell Lines Vary in Lumen Formation (Stem Cell Differentiation) Shaan Ghandi, Unpublished
10 CDX1 Homeobox gene Embryonic intestinal development Postnatal and adult development: restricted to small intestine & colon Regulates intestine-specific genes: Intestinal alkaline phosphatase (IAP), Glu6Pase, A33, MUC2 Often down regulated by methylation in CRC
11 CDX1 is expressed in the differentiated part of the normal crypt CDX1 pab KRT20 mab Expression of CDX1 & KRT20 increase from bottom to top of the crypt in normal colonic mucosa.
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13 A Effects are cumulative and reversible C SW1222 grown 4 weeks in Matrigel Normoxia Hypoxia B Normoxia Hypoxia Replated as single cells then grown for 4 further weeks in Matrigel Normoxia Hypoxia
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16 CDX1: negative feedback on BMI1 HCT116-CDX HCT116-Vec LS174T-Vec LS174T-SiCDX CDX1 Norm Hyp
17 A Norm Hyp B C HCT116-CDX1 CDX1 CDX1 D E HCT116-Vec CDX1 CDX1 F LS174T-Vec LS174T-siCDX1
18 200nM DBZ DBZ 20nM DBZ 0nM DBZ A Norm Notch inhibition induces goblet cell formation in hypoxia Hyp B C
19 I hypoxia
20 Acknowledgements Trevor Yeung Shaan Gandhi Jenny Wilding Neil Ashley Karin Bracht Angie Nicholls Ying Liu Prof Ruth Muschel Prof Adrian Harris Simon Wigfield Ioanna Ledaki Dr Thomas Brunner FACS facility Kevin Clark Craig Waugh Prof Neil Mortensen
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25 A B Dense colony C Lumen colony
26 ' ()*+(,-. /0 0 1()*+,2. ' (3+45. (*+,6*2*. *37,38, 9: );,' (10,< 1==.,> Hypoxia increases probability of symmetric division? &
27 CCK81 LS180 LS513 LS174T RCM1 C99 PCJW C84 HRA19 C80 Gp2d SW1222 SKCO1 SNUC2B SW1417 VACO5 HCA7 WIDR LOVO LS411 HCT15 NCIH716 RKO DLD1 COLO741 CC20 HCT116 SW48 CDX1 CDX1 QMA Vs Affymetrix Group1 Group2 Affymetrix Cell Lines
28 CDX1 and KRT20 protein expression increase post-confluence in LOVO CDX1 KRT20 Tubulin P-con. = post confluence
29
Nature Medicine: doi: /nm.4078
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