Cell line panel drug screening in organoids and 3D systems › - › media › Files › Horizon ›...
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©2019 Horizon Discovery Group Company—All rights reserved. UK Registered Head Office: Building 8100, Cambridge Research Park, Cambridge, CB25 9TL, United Kingdom. UK Registration No. 05363294 | UK VAT No. GB 189 1993 44 Partner logo 1 (if applicable) www.horizondiscovery.com Cell line panel drug screening in organoids and 3D systems Tim Scales, Anett Rada-Kovacs, Helen Pemberton, Ceri Wiggins, Steffen Lawo, Nicola McCarthy, Annette Little and David Sorrell Horizon Discovery Ltd, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, UK 1927 Introduction To meet the need for cell-panel screening in more complex culture systems, we have investigated 3D cell-based screens to more closely model the complex physiological environment found in tumours. Our existing OncoSignature panel of 300 cell lines was evaluated to produce a 200 strong cell line panel which form 3D spheroids in ultra-low attachment (ULA) plates. Using Horizon’s screening platform, we performed compound screens comparing activity in 2D and 3D systems and identified compounds showing differential 2D versus 3D activity. We also extended our 3D screening into organoid cultures. Organoids have increased complexity in both structure and cell heterogeneity compared with spheroids, and have historically been challenging to use in high-throughput screening. However, our proof-of-concept study produced robust data that are in agreement with known genotype based compound sensitivities. Finally, we used our screening platform to evaluate the power of CRISPR- Cas9 approaches in 3D cell-based systems, and are now developing functional genomic screens in organoid models. Cell-panel Screening Platform Development of 3D screen Starting with our 300-strong OncoSignature panel of indication diverse and genetically well-characterised lines, we first evaluated the ability of all cell lines to form spheroids in 384 well ULA plates. Based on imaging, over 200 lines were identified as being spheroid-forming. Example imaging: A549 cell line Indication distribution of the 3D cell line panel The proportion of lines which formed spheroids were representative of the cancer types that make up the 2D panel of solid tumour derived lines. 2D versus 3D HTS Compound Screening Examples of DLD-1 dose response curves for selected agents 2D versus 3D compound screen in DLD-1 cells. Agents are ranked by magnitude of difference between 2D EC 50 and 3D EC 50 values. Dotted lines represent a 3-fold difference in EC 50 between assay formats. Target EGFR, HR2 MEK MEK EGFR IGFR WEE1 PI3K ALK ALK DNA Synthesis DNA Alkylating BRAF HDAC PORN, Wnt Chk1, Chk2 ERK HIF-1 alpha MDM2 Wnt/β-Catenin BCL2 BRAF C-Met, ALK HDAC BRAF PI3K, mTOR DNA (hypoxia) Proteasome BCR-ABL Microtubule Microtubule Auroura mTOR TOPO1 PARP DNA MTAse Agent High-throughput Format Screening in Colorectal Organoids To take 3D cell-based screening beyond cell line based spheroids, we performed a proof-of-concept study using colorectal organoids in a high- throughput format. Historically, organoids have been available in limited quantities and with considerable batch-to-batch variation, but bioprocessing technologies developed by our partner Cellesce have enabled us to overcome these challenges. The two organoid lines showed expected morphology and growth in the assay and the morphology reflected that described for colorectal organoids. We found that the BRAF mutant organoids were exquisitely sensitive to the BRAF inhibitor dabrafenib, which is a response that mirrors clinical data. Gene Isolation A Isolation B KRAS WT G12D BRAF K601E WT EGFR R512K WT Selected genetic characteristics of the two organoid lines Isolation A Isolation B Differing morphology of the two colorectal organoid lines Examples of HTS-format organoid assay dose response curves Conclusion We are developing a 3D cell panel screen using a panel of 200 cell lines suitable for 3D spheroid format drug-profiling. • Agents known to show greater activity in 3D than 2D, such as MEK inhibitors, were identified in drug panel screens and differential 3D/2D activity for these agents were validated in soft-agar assays (data not shown). • Our proof-of-concept study using organoids demonstrated recapitulation of clinical observations in this next-generation 3D model. We have performed a pooled CRISPR-Cas9 screen under 2D/3D conditions and identified BCL2L1 as a sensitiser of cell survival in 3D. • Utilising our expertise in CRISPR-Cas9 screening, we are developing workflows to facilitate screening in organoid systems. CRISPR-Cas9 Pooled Screening in 3D A pooled CRISPR-Cas9 knockout screen was performed in DLD-1 cells using a tumour suppressor library targeting 110 genes. Cells were grown in standard and low-anchorage conditions. In the 2D versus 3D screen, MEK and EGFR inhibitors clearly showed higher activity in 3D than 2D, with the EC 50 being >10-fold more potent in 3D. Example of drug combination dosing (Trametinib x Pictilisib) in both 2D and 3D Dose Matrix | None | 500 | DLD-1 | None | 384 | GM | Z Factor | CTG-2.0 | 72h GDC-0941 (uM) 0 9.8e-4 .0099 .099 1 Trametinib (uM) 0 2.9e-4 3e-3 .03 .3 Growth Inhibition (%) N=10 DT-1399648 - 2 11 5 8 10 25 36 57 9 19 17 15 21 20 33 53 63 11 19 19 12 22 32 38 51 74 14 25 32 29 27 29 42 58 76 26 29 26 30 31 36 48 65 85 34 42 47 40 43 44 62 75 88 51 53 56 54 57 57 72 84 95 59 65 64 64 64 71 80 89 97 74 76 79 77 78 82 88 94 96 2D Dose Matrix | None | 750 | DLD-1 | None | 384 | GM | Z Factor | CTG 3D | 72h GDC-0941 (uM) 0 9.8e-4 .0099 .1 1 Trametinib (uM) 0 2.9e-4 3e-3 .03 .3 Growth Inhibition (%) N=7-10 DT-1399649 - 16 19 10 31 38 61 74 91 15 23 23 21 32 51 69 92 90 22 39 37 43 46 60 74 92 94 52 48 58 60 62 74 81 97 99 73 69 74 76 72 81 88 96 112 81 82 83 78 86 85 95 110 123 81 82 85 88 89 98 106 117 125 92 84 93 95 95 94 104 135 140 90 90 91 95 90 95 111 136 139 3D 2D vs. low anchorage CRISPR-Cas9 screen workflow CRISPR-Cas9 Screening in Organoids Horizon’s screening expertise could also be applied to genetic screening of organoids and we are now developing workflows in this area. The screen revealed BCL2L1 as a major sensitiser of DLD-1 cell survival exclusively in 3D conditions. 2D vs. 3D ranking of lethality by gene Day 1 Day 4 Day 8 To test the ability of our screening platform to differentiate compound activity in 2D versus 3D, growth assays were performed. Example data using the colorectal cancer cell line DLD-1 with CellTiter-Glo2.0™/CellTiter-Glo 3D™ readouts (Promega) are shown.
Transcript of Cell line panel drug screening in organoids and 3D systems › - › media › Files › Horizon ›...
©2019 Horizon Discovery Group Company—All rights reserved. UK Registered Head Office: Building 8100, Cambridge Research Park, Cambridge, CB25 9TL, United Kingdom. UK Registration No. 05363294 | UK VAT No. GB 189 1993 44
Partner logo 1 (if applicable)
www.horizondiscovery.com
Cell line panel drug screening in organoids and 3D systemsTim Scales, Anett Rada-Kovacs, Helen Pemberton, Ceri Wiggins, Steffen Lawo, Nicola McCarthy, Annette Little and David SorrellHorizon Discovery Ltd, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, UK
1927
IntroductionTo meet the need for cell-panel screening in more complex culture systems, we have investigated 3D cell-based screens to more closely model the complex physiological environment found in tumours. Our existing OncoSignature panel of 300 cell lines was evaluated to produce a 200 strong cell line panel which form 3D spheroids in ultra-low attachment (ULA) plates. Using Horizon’s screening platform, we performed compound screens comparing activity in 2D and 3D systems and identified compounds showing differential 2D versus 3D activity.We also extended our 3D screening into organoid cultures. Organoids have increased complexity in both structure and cell heterogeneity compared with spheroids, and have historically been challenging to use in high-throughput screening. However, our proof-of-concept study produced robust data that are in agreement with known genotype based compound sensitivities.Finally, we used our screening platform to evaluate the power of CRISPR-Cas9 approaches in 3D cell-based systems, and are now developing functional genomic screens in organoid models.
Cell-panel Screening Platform
Development of 3D screenStarting with our 300-strong OncoSignature panel of indication diverse and genetically well-characterised lines, we first evaluated the ability of all cell lines to form spheroids in 384 well ULA plates.Based on imaging, over 200 lines were identified as being spheroid-forming.
Example imaging: A549 cell line
Indication distribution of the3D cell line panel
The proportion of lines which formed spheroids were representative of the cancer types that make up the 2D panel of solid tumour derived lines.
2D versus 3D HTS Compound Screening
Examples of DLD-1 dose response curves for selected agents
2D versus 3D compound screen in DLD-1 cells. Agents are ranked by magnitude of difference between 2D EC50 and 3D EC50 values. Dotted lines
represent a 3-fold difference in EC50 between assay formats.
Target
EGFR
, HR
2M
EKM
EKEG
FRIG
FRW
EE1
PI3K
ALK
ALK
DN
A S
ynth
esis
DN
A A
lkyl
atin
gB
RA
FH
DA
C
PORN
, Wnt
Chk1
, Chk
2
ERK
HIF
-1 a
lpha
MD
M2
Wnt
/β-C
aten
in
BCL2
BRA
F
C-M
et, A
LK
HD
AC
BRA
F
PI3K
, mTO
R
DN
A (h
ypox
ia)
Prot
easo
me
BCR-
ABL
Mic
rotu
bule
Mic
rotu
bule
Aur
oura
mTO
R
TOPO
1
PARP
DN
A M
TAse
Agent
High-throughput Format Screening in Colorectal OrganoidsTo take 3D cell-based screening beyond cell line based spheroids, we performed a proof-of-concept study using colorectal organoids in a high-throughput format. Historically, organoids have been available in limited quantities and with considerable batch-to-batch variation, but bioprocessing technologies developed by our partner Cellesce have enabled us to overcome these challenges.
The two organoid lines showed expected morphology and growth in the assay and the morphology reflected that described for colorectal organoids. We found that the BRAF mutant organoids were exquisitely sensitive to the BRAF inhibitor dabrafenib, which is a response that mirrors clinical data.
Gene Isolation A Isolation B
KRAS WT G12D
BRAF K601E WT
EGFR R512K WT
Selected genetic characteristics of the two organoid lines
Isolation A Isolation B
Differing morphology of the two colorectal organoid lines
Examples of HTS-format organoid assay dose response curves
ConclusionWe are developing a 3D cell panel screen using a panel of 200 cell lines suitable for 3D spheroid format drug-profiling.• Agents known to show greater activity in 3D than 2D, such as MEK inhibitors, were identified in drug panel screens and differential 3D/2D activity for these agents were validated in soft-agar assays (data not shown).• Our proof-of-concept study using organoids demonstrated recapitulation of clinical observations in this next-generation 3D model. We have performed a pooled CRISPR-Cas9 screen under 2D/3D conditions and identified BCL2L1 as a sensitiser of cell survival in 3D.• Utilising our expertise in CRISPR-Cas9 screening, we are developing workflows to facilitate screening in organoid systems.
CRISPR-Cas9 Pooled Screening in 3DA pooled CRISPR-Cas9 knockout screen was performed in DLD-1 cells using a tumour suppressor library targeting 110 genes. Cells were grown in standard and low-anchorage conditions.
In the 2D versus 3D screen, MEK and EGFR inhibitors clearly showed higher activity in 3D than 2D, with the EC50 being >10-fold more potent in 3D.
Example of drug combination dosing (Trametinib x Pictilisib) in both 2D and 3D
Dose Matrix | None | 500 | DLD-1 | None | 384 | GM | Z Factor | CTG-2.0 | 72h
GDC-
0941
(uM
)0
9.8e
-4.0
099
.099
1
Trametinib (uM)0 2.9e-4 3e-3 .03 .3
Grow
th In
hibi
tion
(%)
N=
10
DT-1399648
-
2
11
5
8
10
25
36
57
9
19
17
15
21
20
33
53
63
11
19
19
12
22
32
38
51
74
14
25
32
29
27
29
42
58
76
26
29
26
30
31
36
48
65
85
34
42
47
40
43
44
62
75
88
51
53
56
54
57
57
72
84
95
59
65
64
64
64
71
80
89
97
74
76
79
77
78
82
88
94
96
2D
Dose Matrix | None | 750 | DLD-1 | None | 384 | GM | Z Factor | CTG 3D | 72h
GDC-
0941
(uM
)0
9.8e
-4.0
099
.11
Trametinib (uM)0 2.9e-4 3e-3 .03 .3
Grow
th In
hibi
tion
(%)
N=
7-10
DT-1399649
-
16
19
10
31
38
61
74
91
15
23
23
21
32
51
69
92
90
22
39
37
43
46
60
74
92
94
52
48
58
60
62
74
81
97
99
73
69
74
76
72
81
88
96
112
81
82
83
78
86
85
95
110
123
81
82
85
88
89
98
106
117
125
92
84
93
95
95
94
104
135
140
90
90
91
95
90
95
111
136
139
3D
2D vs. low anchorage CRISPR-Cas9 screen workflow
CRISPR-Cas9 Screening in OrganoidsHorizon’s screening expertise could also be applied to genetic screening of organoids and we are now developing workflows in this area.
The screen revealed BCL2L1 as a major sensitiser of DLD-1 cell survival exclusively in 3D conditions.
2D vs. 3D ranking of lethality by gene
Day 1
Day 4
Day 8
To test the ability of our screening platform to differentiate compound activity in 2D versus 3D, growth assays were performed. Example data using the colorectal cancer cell line DLD-1 with CellTiter-Glo2.0™/CellTiter-Glo 3D™ readouts (Promega) are shown.
