Multicellular 3D Models for Preclinical Drug Discovery...Isotypes α-SMA-APC Isotypes α -SMA APC C5 6 Mono M φ GM-CSF M φ M-CSF ated c W C-H 0 65536 196608 0 65536 131072 196608

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


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


5

Overview on 3D models

➢ Many more ….. Soft agar, 3D-printing…..etc.

➢ Appropriate assay strongly depends on focus of interest


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


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)


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


9

Creating a cellular model

1. Define markers for detection

2. Titration of suitable cell-cell-ratios

3. Model phenotypes


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


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%


PE-A

AP

C-A

-101

102

103

104

105

-101

102

103

104

105 100,00% 0,00%

0,00% 0,00%


PE-A

AP

C-A

-10110

210

310

410

5

-102

102

103

104

105 0,00% 0,09%

99,91% 0,00%


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%


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


FSC-W

FS

C-H

0 65536 1966080

65536

131072

196608

262144

singlets


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


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%


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%


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%


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%


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



14


PE-A

AP

C-A

-101

102

103

104

105

-102

103

104

105 99,28% 0,00%

0,72% 0,00%


PE-A

AP

C-A

-101

102

103

104

105

-102

-101

102

103

104

105 0,00% 0,00%

32,36% 67,64%


PE-A

AP

C-A

-101

102

103

104

105

-102

-101

102

103

104

105 0,00% 0,00%

99,85% 0,15%


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%


PE-A

AP

C-A

-101

102

103

104

105

-102

102

103

104

105 92,80% 0,28%

6,93% 0,00%


PE-A

AP

C-A

-101

102

103

104

105

-102

-101

102

103

104

105 0,00% 0,00%

99,71% 0,29%


PE-A

AP

C-A

-102

102

103

104

105

-102

-101

102

103

104

105 0,00% 0,00%

0,74% 99,26%


PE-A

AP

C-A

-101

102

103

104

105

-102

-101

102

103

104

105 0,00% 0,00%

99,63% 0,37%


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%


PE-A

AP

C-A

-101

102

103

104

105

-102

102

103

104

105 22,03% 0,00%

77,97% 0,00%


PE-A

AP

C-A

-101

102

103

104

105

-101

102

103

104

105 0,00% 0,00%

100,00% 0,00%


PE-A

AP

C-A

-101

102

103

104

105

-102

-101

102

103

104

105 0,00% 0,00%

100,00% 0,00%


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%


PE-A

AP

C-A

-101

102

103

104

105

-102

-101

102

103

104

105

0,00%51,44%

0,00%48,56%


PE-A

AP

C-A

-101

102

103

104

105

-101

102

103

104

105

0,00%100,00%

0,00%0,00%


PE-A

AP

C-A

-101

102

103

104

105

-102

-101

102

103

104

105

100,00%0,00%

0,00%0,00%


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%


PE-A

AP

C-A

-101

102

103

104

105

-102

-101

102

103

104

105

0,00%17,36%

0,18%82,45%


PE-A

AP

C-A

101

102

103

104

105

-102

102

103

104

105

0,05%99,71%

0,05%0,19%


PE-A

AP

C-A

101

102

103

104

105

-102

102

103

104

105

0,00%99,80%

0,05%0,15%


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+



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%


Alexa Fluor 488-AP

E-A

-102

102

103

104

105

-103

-102

103

104

105

84,58%12,35%

2,79%0,28%


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%


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%


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%


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%


PE-A

AP

C-A

-10210

110

210

310

410

5

-101

102

103

104

105

1,35%98,63%

0,02%0,00%


PE-A

AP

C-A

-101

102

103

104

105

-102

-101

102

103

104

105

0,00%49,65%

0,00%50,35%


PE-A

AP

C-A

-101

102

103

104

105

-101

102

103

104

105

39,32%60,68%

0,00%0,00%


PE-A

AP

C-A

-101

102

103

104

105

-101

102

103

104

105

0,02%99,98%

0,00%0,00%


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%


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%


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%


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%


Alexa Fluor 488-A

PE

-A

-101

102

103

104

105

-102

102

103

104

105

0,14% 98,63%

0,22% 1,01%


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%


Alexa Fluor 488-A

PE

-A

-101

102

103

104

105

-101

102

103

104

105

0,00% 0,17%

15,91% 83,92%


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%


Alexa Fluor 488-A

PE

-A

101

102

103

104

105

-102

102

103

104

105

0,00% 0,00%

15,70% 84,30%


Alexa Fluor 488-A

PE

-A

101

102

103

104

105

-102

102

103

104

105

0,12% 31,37%

15,09% 53,42%


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



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


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


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


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



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


➢ 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


Conclusion


➢ 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


➢ 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


Miriam Ehmeier

Stefan Wenzel

Felix Neumann

Melanie Baumgärtner


Multicellular 3D Models for Preclinical Drug Discovery...Isotypes α-SMA-APC Isotypes α -SMA APC C5 6 Mono M φ GM-CSF M φ M-CSF ated c W C-H 0 65536 196608 0 65536 131072 196608

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