Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology,
Lab Head Cancer Stem Cells
Design of microenvironment components to benefit combination treatments
Multicellular 3D Models for Preclinical Drug Discovery
2
▪ CSC are tumor initiating cells
▪ CSC share common features with normal stem cells
Cancer Stem Cells – expansion and homeostasis
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
3
Cancer Stem Cells are responsible for resistance and metastasis
Inconsistent/ inconclusive research result
▪ Cancer stem cells as well as other cells of the tumor microenvironmentcan shape their phenotype upon interaction with other cells or solublefactors
▪ Tumor plasticity is mainly governed by the tumor microenvironment andstemness functions
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
4
The microenvironment governs tumors plasticity
2
1 Tumor microenvironemnt & CSC-niche
▪ Made by tumor infiltrating cells
▪ Hypoxic
▪ Induces stemness by direct interaction with orby factors produced by niche cells
▪ Enables survival, migration and metastasis ofCSC
Targeting Tumorplasticity
▪ Extracellular targets are good to target
▪ Intracellular and nuclear targets are not easy totarget
▪ Targeting interactions with niche cells or nichefactors (ECM) influence intracellular and nucleartargets
Cancer stem cell
Immune cells
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
5
Overview on 3D models
➢ Many more ….. Soft agar, 3D-printing…..etc.
➢ Appropriate assay strongly depends on focus of interest
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
6
Simplistic modelling of Tumor features
✓ Spheres can be generated from primary cell cultures w/o addition of cytokines
✓ These spheres generate ECM and secrete tumor-relevant cytokines themselves
✓ A label retaining population is maintained in our sphere cultures/ CSC are enriched
Susanne Wendland
Sandra Müller
Cfse (proliferation)
Also proven on protein level
Fibronectin p27 p21WAF
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
stemness
abc
7
Modelling cellular complexity of a tumor
➢ Modulation of sphere formation
➢ via co-culturing
➢ Addition of ECM, growth factors, cytokines Monocytes
Macrophages
Fibroblasts
Mesenchymal stem cellsInterfering with
b)Cellular components (stroma)
c)Cellular components (innate immune cells)
a)Composition (ECM/ soluble factors)
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
8
Stemness in a cancer context
Cancer Stem Cells can self renew, are resistant to apoptosis and conventionaltherapy and evade the immune system.
CSC can undergo EMT (epithelial mesenchymal transition). EMT generates amesenchymal like cell. CSC can also switch back by MET and colonize therebydistant metastases.
Adapted from Drasin et al, Breast cancer research 2011 13:226 Brabletz et al, Cancer Cell 2012 22:Dec 11
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
9
Creating a cellular model
1. Define markers for detection
2. Titration of suitable cell-cell-ratios
3. Model phenotypes
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
10
NSCLC primary spheres
sp
here
s/
well [
% t
o c
on
tro
l]
0 1:1 1:3 1:5 1:1 1:3 1:5 1:1 1:3 1:50
100
200
300
400
500
#Pry006n=4
Pry Pry+M-CSF M #n=2 Pry+GM-CSF M #n=1 Pry+ Mono #n=3
*
CrC primary spheres
sp
here
s/
well [
% t
o c
on
tro
l]
0 1:1 1:3 1:5 1:1 1:3 1:5 1:1 1:3 1:50
200
400
600
800
#Pry007n=4
Pry Pry+M-CSF M #n=2 Pry+GM-CSF M #n=1 Pry+Mono #n=3
*
PrC primary spheres
sp
here
s/
well [
% t
o c
on
tro
l]
0 1:1 1:3 1:5 1:1 1:3 1:5 1:1 1:3 1:50
500
1000
1500
#Pry076n=4
Pry Pry+M-CSF M #n=1 Pry+GM-CSF M #n=1 Pry+Mono #n=3
*
Susanne Wendland
Sandra Müller, Jasmin Meckler
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
Co-culturing with cells of the tumor microenvironment enhance sphere formation (marker of stemness)
Fib
robla
sts
Myelo
idCells
Myelo
idCells
CoCultures Monocytes_Tube_022_022.fcs compensated
Alexa Fluor 488-AC
ount
-103
-102
103
104
105
0
1
2
2
3
M110,84%
M266,76%
M319,80%
CoCultures Pry and Pry fibro_Tube_040_040.fcs compensated
Alexa Fluor 488-A
Count
100
102
103
104
105
0
1
2
3
4
M19,49%
M266,99%
M321,47%
CoCultures Monocytes_Tube_022_022.fcs compensated
Alexa Fluor 488-A
Count
101
102
103
104
105
0
1
2
4
5
M10,38%
M263,23%
M335,00%
CoCultures Pry and Pry fibro_Tube_046_046.fcs compensated
Alexa Fluor 488-A
Count
-101
102
103
104
105
0
2
4
7
9
M17,71%
M277,45%
M313,17%
CoCultures Pry fibro mono_Tube_031_031.fcs compensated
Alexa Fluor 488-A
Count
-103-10
210
310
410
5
0
1
3
4
5
M11,91%
M260,07%
M333,79%
10000 stromal cells(fibroblasts)
Ratio 1:2
10000 stromal cells(fibroblasts)
10000 Myeloid cellsRatio 1:2
pool pool
➢ M2 macrophages enhance label retaining population (slowproliferating)
➢ Fibroblasts decrease the label retaining population
FiMoMφMo Fi
Sandra Müller
11 Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
M-CSF
Effect of tumor microenvironment components on sphere forming cells
5000 Tumor cells
12
Detection of stromal components is difficult due to a high plasticity
M-CSF 1605 male_C2_C02_018.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-101
102
103
104
105 0,00% 0,71%
99,29% 0,00%
M-CSF 1605 male_C3_C03_019.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-101
102
103
104
105 100,00% 0,00%
0,00% 0,00%
GM-CSF 1605 male_D2_D02_026.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105 0,00% 0,57%
98,29% 1,14%
GM-CSF 1605 male_D3_D03_027.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-101
102
103
104
105 98,47% 0,00%
1,53% 0,00%
Mono 1606 female D0_B2_B02_010.fcs compensated
PE-A
AP
C-A
-10110
210
310
410
5
-102
102
103
104
105 0,00% 0,00%
100,00% 0,00%
Mono 1606 female D0_B3_B03_011.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-101
102
103
104
105 100,00% 0,00%
0,00% 0,00%
Mono 1606 female D0_B2_B02_010.fcs compensated
PE-A
AP
C-A
-10110
210
310
410
5
-102
102
103
104
105 0,00% 0,09%
99,91% 0,00%
Mono 1606 female D0_B3_B03_011.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-101
102
103
104
105 99,57% 0,00%
0,43% 0,00%
Pry006 CSFE_E2_E02_034.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105 0,00% 0,25%
95,81% 3,94%
Pry006 CSFE_E3_E03_035.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-101
102
103
104
105 69,53% 1,48%
28,53% 0,46%
MRC-5_A2_A02_002.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105 0,00% 2,13%
95,74% 2,13%
MRC-5_A3_A03_003.fcs compensated
PE-A
AP
C-A
100
102
103
104
105
-101
102
103
104
105 96,05% 0,00%
3,95% 0,00%
α-SMA-APCIsotypes
α-SMA-APCIsotypes
MRC5
Pry
006
Mono
Mφ
GM
-CSF
Mφ
M-C
SF
Dendritic
Mono 1606 female D0_B1_B01_009.fcs compensated
FSC-W
FS
C-H
0 65536 1966080
65536
131072
196608
262144
singlets
Mono 1606 female D0_B1_B01_009.fcs compensated
FSC-A
SS
C-A
0 65536 131072 196608 2621440
65536
131072
196608
262144
viable mono
dendritic
Α-SMA clone #E184Also stains myeloid cells
αSM
A A
PC
PE
#1606 f
Stefanie Scharrelmann
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
surface_Tube_004_004.fcs compensated
APC-A
Ale
xa
Flu
or
70
0-A
-10210
110
310
410
5
-101
102
103
104
105 0,00% 0,16%
32,31% 67,54%
surface_Tube_005_005.fcs compensated
PE-A
Ale
xa
Flu
or
70
0-A
-101
102
103
104
105
-101
102
103
104
105
0,02%99,86%
0,00%0,12%
surface_Tube_006_006.fcs compensated
PE-A
Ale
xa
Flu
or
70
0-A
-101
102
103
104
105
-101
102
103
104
105
45,29%53,83%
0,80%0,07%
surface_Tube_007_007.fcs compensated
PE-A
Ale
xa
Flu
or
70
0-A
-101
102
103
104
105
-101
102
103
104
105
0,00%0,66%
0,02%99,33%
surface_Tube_008_008.fcs compensated
PE-A
Ale
xa
Flu
or
70
0-A
-101
102
103
104
105
-101
102
103
104
105
0,05%98,74%
0,11%1,10%
CD140a-PE CD140b-APC CD31-PE CD45-AF700 EPCAM-PE
13
Also PDGFRβ and other markers are expressed by myeloid cells
High stromal plasticity, high plasticity of myeloid cells, tumor plasticity……
…. Singular targeting is complicated and combinations are a must
microenvironment
cancer bulk
cancer stem cell (TiC)
immune system
Stefanie Scharrelmann
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
14
MRC-5_A4_A04_004.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
103
104
105 99,28% 0,00%
0,72% 0,00%
MRC-5_A5_A05_005.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105 0,00% 0,00%
32,36% 67,64%
MRC-5_A6_A06_006.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105 0,00% 0,00%
99,85% 0,15%
MRC-5_A8_A08_008.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105 0,00% 0,00%
100,00% 0,00%
Pry006-CSFE_A2_A02_002.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105 0,17% 0,08%
98,92% 0,83%
Pry006-CSFE_A4_A04_004.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
102
103
104
105 92,80% 0,28%
6,93% 0,00%
Pry006-CSFE_A5_A05_005.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105 0,00% 0,00%
99,71% 0,29%
Pry006-CSFE_A6_A06_006.fcs compensated
PE-A
AP
C-A
-102
102
103
104
105
-102
-101
102
103
104
105 0,00% 0,00%
0,74% 99,26%
Pry006-CSFE_A8_A08_008.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105 0,00% 0,00%
99,63% 0,37%
MRC-5_A2_A02_002.fcs compensated
PE-A
AP
C-A
-102
102
103
104
105
-102
100
102
103
104
105 0,00% 0,10%
99,85% 0,05%
Mono 1606 female d0_C2_C02_017.fcs compensated
PE-A
AP
C-A
-102
102
103
104
105
-102
-101
102
103
104
105 0,23% 0,23%
98,98% 0,57%
Mono 1606 female d0_C4_C04_019.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
102
103
104
105 22,03% 0,00%
77,97% 0,00%
Mono 1606 female d0_C5_C05_020.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-101
102
103
104
105 0,00% 0,00%
100,00% 0,00%
Mono 1606 female d0_C6_C06_021.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105 0,00% 0,00%
100,00% 0,00%
Mono 1606 female d0_C8_C08_023.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-101
102
103
104
105 0,00% 0,00%
1,36% 98,64%
CD140b-APC CD140a-PE EPCAM-PEIsotypes CD45-PE
MRC5
Pry
006
Mono
Mφ
GM
-CSF
Mφ
M-C
SF
GM-CSF Makro d1606_C2_C02_011.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105
0,00%99,57%
0,00%0,43%
GM-CSF Makro d1606_C4_C04_013.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105
0,00%51,44%
0,00%48,56%
GM-CSF Makro d1606_C5_C05_014.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-101
102
103
104
105
0,00%100,00%
0,00%0,00%
GM-CSF Makro d1606_C9_C09_018.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105
100,00%0,00%
0,00%0,00%
GM-CSF Makro d1606_C7_C07_016.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105
0,21%99,79%
0,00%0,00%
M-CSF Makro d1606_E2_E02_027.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-101
102
103
104
105
0,82%97,82%
0,55%0,82%
M-CSF Makro d1606_E4_E04_029.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105
0,00%17,36%
0,18%82,45%
M-CSF Makro d1606_E5_E05_030.fcs compensated
PE-A
AP
C-A
101
102
103
104
105
-102
102
103
104
105
0,05%99,71%
0,05%0,19%
M-CSF Makro d1606_E7_E07_032.fcs compensated
PE-A
AP
C-A
101
102
103
104
105
-102
102
103
104
105
0,00%99,80%
0,05%0,15%
M-CSF Makro d1606_E9_E09_034.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105
99,24%0,29%
0,48%0,00%
14
Definition of microenvironment components
Fibroblasts: PDGFRα+/PDGFRβ+/EPCAM-/CD45-
Tumor:
PDGFRα-/PDGFRβ+/EPCAM+/CD45-
Myeloid cells:
PDGFRα-/PDGFRβ+/EPCAM-/CD45+
Stefanie Scharrelmann
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
CD140b-APC CD140a-PE EPCAM-PEIsotypes CD45-PE
MRC5
Pry
006
Tu+
Fib
roTu-
Fib
ro+
Mono
15
Co-culture analyses
Fibroblasts: not detectable
Tumor: EPCAM+/CFSE
Myeloid cells: CD45+
Pry006 CSFE_A2_A02_002.fcs compensated
PE-A
Ale
xa F
luor
700-A
-103-10
210
310
410
5
-102
102
103
104
105
2,97%96,90%
0,09%0,03%
Pry006 CSFE_A2_A02_002.fcs compensated
Alexa Fluor 488-AP
E-A
-102
102
103
104
105
-103
-102
103
104
105
84,58%12,35%
2,79%0,28%
Pry006 CSFE_A4_A04_004.fcs compensated
Alexa Fluor 488-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105
67,80%7,09%
23,30%1,81%
Pry006 CSFE_A7_A07_007.fcs compensated
Alexa Fluor 488-A
PE
-A
-101
102
103
104
105
-102
100
102
103
104
105
0,65%1,23%
97,57%0,55%
Pry006 CSFE_A9_A09_009.fcs compensated
PE-A
Ale
xa F
luor
700-A
-101
102
103
104
105
-102
-101
102
103
104
105
0,00%100,00%
0,00%0,00%
Pry006 CSFE_A5_A05_005.fcs compensated
Alexa Fluor 488-A
PE
-A
-101
102
103
104
105
-101
102
103
104
105
90,52%9,48%
0,00%0,00%
MRC5 p11_C2_C02_018.fcs compensated
PE-A
Ale
xa F
luor
700-A
-10210
110
210
310
410
5
-102
-101
102
103
104
105
1,35%98,63%
0,02%0,00%
MRC5 p11_C2_C02_018.fcs compensated
PE-A
AP
C-A
-10210
110
210
310
410
5
-101
102
103
104
105
1,35%98,63%
0,02%0,00%
MRC5 p11_C4_C04_020.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105
0,00%49,65%
0,00%50,35%
MRC5 p11_C5_C05_021.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-101
102
103
104
105
39,32%60,68%
0,00%0,00%
MRC5 p11_C7_C07_023.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-101
102
103
104
105
0,02%99,98%
0,00%0,00%
MRC5 p11_C9_C09_025.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105
0,00%100,00%
0,00%0,00%
Pry006 CSFE + MRC5_E2_E02_034.fcs compensated
PE-A
AP
C-A
-10210
010
210
310
410
5
-101
102
103
104
105
0,76%99,24%
0,00%0,00%
Pry006 CSFE + MRC5_E2_E02_034.fcs compensated
Alexa Fluor 488-A
PE
-A
-101
102
103
104
105
-102
-101
102
103
104
105
94,51%4,58%
0,91%0,00%
Pry006 CSFE + MRC5_E4_E04_036.fcs compensated
Alexa Fluor 488-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105
1,00% 59,98%
2,33% 36,69%
Pry006 CSFE + MRC5_E5_E05_037.fcs compensated
Alexa Fluor 488-A
PE
-A-10
110
210
310
410
5
-101
102
103
104
105
0,00% 0,03%
3,82% 96,15%
Pry006 CSFE + MRC5_E7_E07_039.fcs compensated
Alexa Fluor 488-A
PE
-A
-101
102
103
104
105
-102
102
103
104
105
0,14% 98,63%
0,22% 1,01%
Pry006 CSFE + MRC5_E9_E09_041.fcs compensated
Alexa Fluor 488-A
PE
-A
-101
102
103
104
105
-101
102
103
104
105
0,00% 0,00%
4,42% 95,58%
Mono 1606 + Pry006 CSFE + MRC5_G2_G02_050.fcs compensated
PE-A
AP
C-A
-101
102
103
104
105
-102
-101
102
103
104
105
0,02% 0,10%
99,68% 0,21%
Mono 1606 + Pry006 CSFE + MRC5_G2_G02_050.fcs compensated
Alexa Fluor 488-A
PE
-A
-101
102
103
104
105
-101
102
103
104
105
0,00% 0,17%
15,91% 83,92%
Mono 1606 + Pry006 CSFE + MRC5_G4_G04_052.fcs compensated
Alexa Fluor 488-A
AP
C-A
101
102
103
104
105
-102
102
103
104
105
6,13% 54,43%
8,24% 31,20%
Mono 1606 + Pry006 CSFE + MRC5_G5_G05_053.fcs compensated
Alexa Fluor 488-A
PE
-A
101
102
103
104
105
-102
102
103
104
105
0,00% 0,00%
15,70% 84,30%
Mono 1606 + Pry006 CSFE + MRC5_G7_G07_055.fcs compensated
Alexa Fluor 488-A
PE
-A
101
102
103
104
105
-102
102
103
104
105
0,12% 31,37%
15,09% 53,42%
Mono 1606 + Pry006 CSFE + MRC5_G9_G09_057.fcs compensated
Alexa Fluor 488-A
PE
-A
101
102
103
104
105
-102
102
103
104
105
2,10% 41,27%
8,77% 47,86%
#1606 m
Stefanie Scharrelmann
15 Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
16
3D co-culture models to screen novel drugs
• Use model for drug discovery/ drug screening
• Focus on myeloid co-cultures
• Fibroblast detection still challenging
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
Pry076 sph pl14 + 4_G3_G03_027.fcs compensated
Alexa Fluor 488-A
PE
-A
102
103
104
105
-101
102
103
104
105
99,99%0,01%
0,00%0,00%
Pry007 sph pl3 + 23_E3_E03_019.fcs compensated
Alexa Fluor 488-A
PE
-A
100
102
103
104
105
-101
102
103
104
105
99,81%0,15%
0,04%0,00%
Target expression in primary patientmaterial derived CSC linesPry006 adh p25 + 6_A3_A03_003.fcs compensated
Alexa Fluor 488-A
PE
-A
-101
102
103
104
105
-101
102
103
104
105
0,00% 0,06%
0,04% 99,90%
Pry006 sph pl 25 + 4_B3_B03_007.fcs compensated
Alexa Fluor 488-A
PE
-A
-101
102
103
104
105
-101
102
103
104
105
0,00% 0,09%
0,04% 99,87%
79073835
Pry007 adh p3 + 37_D3_D03_015.fcs compensated
Alexa Fluor 488-A
PE
-A
101
102
103
104
105
-101
102
103
104
105
0,00% 0,01%
0,01% 99,97%
20673140
Pry076 adh p14 + 6_F3_F03_023.fcs compensated
Alexa Fluor 488-A
PE
-A
101
102
103
104
105
-102
-101
102
103
104
105
99,96%0,01%
0,03%0,00%
33613372
CrC
NSCLC
PrC
➢ Expressed in adherent state and sphere state
➢ Equally expressed on myeloid cells
➢ Interaction of tumor cells with myeloid cells
Susanne Wendland
17 Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
Pry006 sph + #2239 m-csf_Tube_006_006.fcs compensated
APC-A
PE
-A
-101
102
103
104
105
-101
102
103
104
105
66,25%5,19%
27,44%1,11%
1383
1509 NSCLC
M-C
SF M
Φ
Inhibition of Interaction of Myeloid cells and Tumor Cells
NSCLC primary spheres
sp
here
s/
well [
% t
o c
on
tro
l]
0 0,5 5 0 0,5 1,5 5 0 0,5 5 0 0,5 1,5 5 0 0,5 5 0 0,5 1,5 5 0 0,5 5 0 0,5 1,5 50
50
100
150
200
#Pry006n=4
HEL mAb HEL mAb HEL mAb HEL mAb
Pry Pry+M-CSF M #n=1 Pry+GM-CSF M #n=1 Pry+ Mono #n=3
NSCLC primary spheres
via
bilit
y [
% t
o c
on
tro
l]
0 0,5 5 0 0,5 1,5 5 0 0,5 5 0 0,5 1,5 5 0 0,5 5 0 0,5 1,5 5 0 0,5 5 0 0,5 1,5 50
50
100
150
#Pry006n=4
HEL mAb HEL mAb HEL mAb HEL mAb
Pry Pry+M-CSF M #n=1 Pry+GM-CSF M #n=1 Pry+ Mono #n=3
➢ Reduction of stemness → reduced
sphere formation
➢ Viability partially influenced, but mainly intact
➢ MoA is independent of “tumor cell killing”
➢ Suitable for combination with standard of care or targeted therapies
microenvironment
cancer bulk
cancer stem cell (TiC)
immune system
Susanne Wendland
18 Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
Conclusion
19 Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
➢ The sphere assay detects stemness → only CSC survive under non adherent conditions
➢ Sphere formation is a function of CSC/TiC
➢ Modification of the assay by adding cellular components of the microenvironment
* showed monocytes and macrophages survive in sphere cocultures
* led to enhanced sphere formation
* for some combinations it also enlarged the label retaining population (macrophages) (dormant CSC)
➢ Fibroblasts were undetectable → further investigation needed to define wether plasticity plays a role
➢ Sphere cocultures with myeloid cells are suitable to investigate specific targets that are part of tumor cell/
myeloid cell interaction → further investigation of combination therapies possible
20
Liver metastases in NOD/SCID NSCLC model
meta
sta
ses/lu
ng
Tu TuFi TuMo TuFiMo Fi Mo FiMo0
1
2
3
4
5
2/10 1/10 3/10 1/10
Lung metastases in NOD/SCID NSCLC model
meta
sta
ses/lu
ng
Tu TuFi TuMo TuFiMo Fi Mo FiMo0
5
10
15
20
25*
unpaired t test w Welch's correction
Outlook
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017
➢ Myeloid cells support 3D cultures and stemness in vitro
➢ Inhibition of the interaction of tumor cells and myeloid cells decreases stemness in vitro
➢ Are stromal cells necessary for the development of a comprehensive 3D model?
• Fibroblasts contribute to a relevant cancer phenotype
• Potentially through cytokines: e.g. IL8, recruitment of myeloidcells to the tumor site
• Colonization of the metastaticniche
• Production of ECM
➢ Further investigation needed to fully understand the role and plasticity of stromal cells in the cancer context
➢ Targets that influence of inhibit the interaction with stromal cells open a new target space
21
ACKNOWLEDGEMENTS
Andree Blaukat
Frank Zenke
Tobias Fischer
Sabine Raab
Richard Schneider
Claudia Wilm
Susanne Wendland
Sandra Müller
Stefanie Scharrelmann
Miriam Ehmeier
Stefan Wenzel
Felix Neumann
Melanie Baumgärtner
Anita Seshire, Merck Biopharma R&D, TiP Oncology, Cellular Pharmacology | 16th Nov 2017