3DModels_for_EVs_Munich_2019_v2_IN

pelobiotech

Studying

Intercellular Communication

by mean of

Extracellular Vesicles in 3D Models

„Optimized Cancer Treatments with 3D Models“

PeloBiotech and ABCbiopply workshop

Irina Nazarenko

Medical Center University of Freiburg

27th March 2019


Progressing tumor attracts and re-programs

stromal and immune cells

Santi et al, Proteomics, 2017

2 · April 4, 2019


Tumor releases into microenvironment variety of

different factors

miRNA

mRNA

3 · 4. April 2019

Schwarzenbach et al., Nature Review 2011


4 · April 4, 2019

Emerging complexity of communication mechanisms


Publications frequency of studies investigating

EVs in cancer

Keywords used for PubMed search

ISEV was founded

in 2011 (Paris, France)

2011 2012 2013 2014 2015 2016 2017 2018

5 · April 4, 2019


Relevance of extracellullar vesicles for liquid biopsy

Modifed - from Stella Kourembanas, Annu. Rev. Physiol.

2015. 77:13–27.

6 · April 4, 2019


Versatile EV functions in cancer

Kogure et al., Journal of Biomedical Science; 2019

7 · April 4, 2019


Emerging diversity of extracellular vesicles

EV

Exosomes

50-150 nm

Microvesicles

120-500 nm

Large

Oncosomes

500-800 nm

Apoptotic

Bodies

0.5-4 µm

8 · April 4, 2019


Biogenesis of extracellular vesicles: microvesicles and exosomes

exosomes

microvesicles

Van Niel and Raposo, Nature Review 2017

9 · April 4, 2019


Proteomics analysis of small EVs (mostly exosomes) and large EVs (mostly

microvesicles and oncosomes) show high discrepancy in protein content

Micianchi et al., Oncotarget 2015

10 · April 4, 2019


The need of physiological in vitro models

in vitro

in vivo

www.yogabox.de

11 · April 4, 2019


Development of a 3D model

for EV production and analysis

Requirements for EV production:

‣ 3D environment in serum-free, or EV-free culture medium

‣ Control of cell viability, proliferation, apoptosis and necrosis

‣ High amount of EVs produced

‣ Efficient and easy recovery of EVs from the 3D cell culture

‣ Possibility to upscale the approach

Requirements for modelling of tumor microenvironment

‣ Easy analysis – microscopy, immunohistochemistry

‣ Possibility of co-culture with stroma cells and other

components of tumor microenvironment

12 · April 4, 2019


Application of 3DCoSeedis TM

to produce and study EVs in 3D environment

Models:

Prostate cancer

Breast cancer

Gastrointestinal cancer

13 · April 4, 2019


Separation of different EVs subtypes from 2D and 3D cultures

Cell Culture Supernatant

depletion of cell debries

800 x g and 1000 x g

Centrifugation: 5000 x g for 45 min at 4°C

EV5

large oncosomes/

apoptotic bodies

Centrifugation: 12000 x g for 45 min at 4°C

EV12

microvesicles

• Ultracentrifugation: 120000 x g for 2 h at 4°C

• PEG / or other reagents-based precipitation

• Ultrafiltration

EV120

exosomes and

other small EVs

Serial filtration steps to remove residual EV

fc fraction

Krafft et al., Nanomedicine 2016

Klump et al., Nanomedicine 2017


Application of 3DCoSeedis TM

to produce and study EVs in 3D environment

Models:

Prostate cancer

Breast cancer

Gastrointestinal cancer

15 · April 4, 2019


Establishment of CWR22-RV1 cells

3D culture conditions

1 day,

FSC

2 day,

FSC

1 day 2 day 3 day 4 day 5 day

Scale bar: 50µm

Input

• 10^6 cells/well

• 5% (EV-depleted) FCS

• 1 matrix= 950 microwells

• 2 matrixes = 16 ml

Liliia Paniushkina

TRAIN-EV

ITN-Marie Curie


Comparative analysis of different EV populations from cells

derived from 2D and 3D conditions

Working steps

2D

(225ml)

3D

(16ml)

Cell

Cult

ure

Sup

erna

tant

Differential centrifugation

EV5 EV12 EV120

OptiPrep density gradient

10

fractions

10

fractions

10

fractions

NTA, microBCA, DLS,

qNano analysis


Cells produce more EVs under 3D condtitions

***

*** ***

1


EV populations produced under 2D and 3D conditions differ in

their distribution among Optiprep gradient fractions

ug/ul

particles/ml

fractions


qNano measurements of EV5 and EV12

Optiprep fractions from 2D and 3D cultures

EV5_2D,3D

EV12_2D

EV12_3D

NP800

Size range: 250-1200nm

EV5 3D fraction contains

large EVs 800-900 nm

NP400

Size range: 250-1000nm

NP400

Size range: 150-1100nm

EV12 3D fraction is more

heterogeneous as 2D

fraction


qNano measurements of EV120

Optiprep fractions from 2D and 3D cultures

EV120_2D

EV120_3D

EV120_2D,

3D

NP100

Size range: 95-

190nm

NP100

Size range: 75-250nm

NP100

Size range: 150-1100nm

EV120 particles from 3D

are smaller


Size distribution of EV5,12,120 vesicles

from 2D and 3D cultures


To sum up

‣ The 3DCoSeedis TM are applicable for the EV production in a prostate cancer

model

‣ 3D conditions differentially affect EV population distribution

23 · April 4, 2019


Application of 3DCoSeedis TM

to produce and study EVs in 3D environment

Models:

Prostate cancer

Breast cancer

Richa Khanduri

Gastrointestinal cancer

24 · April 4, 2019


Studying breast cancer small EVs

Cel line

MDA-MB-231 BT-549 MCF7 MDA-MB-361

Molecular

Classification

Triple negative

(Claudin low)

Triple negative

(Claudin low)

Luminal A

Luminal B

Tumor Type Adenocarcinoma Invasive ductal

carcinoma

Adenocarcinoma

Adenocarcinoma

Source

Metastasis,

Pleural Effusion

Primary tumor

Metastasis, Pleural

Effusion

Metastasis, Brain

Phenotype

Mesenchymal

(Post-EMT)

Mesenchymal

(Post-EMT)

Epithelial

Epithelial

Receptor

Expression

ER-, PR-

HER2 low , EGFR+,

ER-, PR-

HER2-, EGFR+

ER+, PR+/-,

HER2 low , EGFR low

ER+, PR+/-

HER2+, EGFR+

Tspan8

Expression

TSPAN8 -/low Tspan8- Tspan8- Tspan8+

25 · April 4, 2019


MDA-MB-231

231-Tspan8

Morphology of cells,

growing under

2D conditons

BT-549

BT-Tspan8

MCF7

MCF7-

Tspan8

26 · April 4, 2019


Establishment of the 3D culture for EV production

MDA-MB-231

MDA-MB-231Tspan8

Day 1 Day 3 Day 5 Day 7 Day 1 Day 3 Day 5 Day 7

MCF7

MCF7-Tspan8

Day 1 Day 3 Day 5 Day 7 Day 1 Day 3 Day 5 Day 7

1000 cells

per microwell

400 cells

per microwell

100 cells

per

microwell

1000 cells

per microwell

400 cells

per microwell

100 cells

per microwell

27 · April 4, 2019


Establishment of the 3D culture for EV production

MDA-MB-231

MDA-MB-231Tspan8

Day 1 Day 3 Day 5 Day 7 Day 1 Day 3 Day 5 Day 7

MCF7

MCF7-Tspan8

Day 1 Day 3 Day 5 Day 7 Day 1 Day 3 Day 5 Day 7

No FBS

FBS Withdrawal

FBS

No FBS

FBS Withdrawal

FBS

28 · April 4, 2019


Establishment of the 3D culture for EV production

MDA-MB-231

MDA-MB-231Tspan8

Day 1 Day 3 Day 5 Day 7 Day 1 Day 3 Day 5 Day 7

MCF7

MCF7-Tspan8

Day 1 Day 3 Day 5 Day 7 Day 1 Day 3 Day 5 Day 7

No FBS

FBS

Withdrawal

FBS

No FBS

FBS

Withdrawal

FBS

29 · April 4, 2019


Establishment of the 3D culture for EV production

MDA-MB-361

2000 cells

per microwell

Day 1 Day 3 Day 5 Day 7

MDA-MB-231

1500 cells

per microwell

231-Tspan8

1500 cells

per microwell

BT-549

1500 cells

per microwell

BT-Tspan8

1500 cells

per microwell

MCF7

2000 cells

per microwell

30 · April 4, 2019

MCF7-

Tspan8

2000 cells

per microwell


Immunohistochemistry of breast cancer cell aggregates

Day 1 Day 3 Day 5 Day 7

MDA-MB-231

231-Tspan8

MCF7

MCF7-

Tspan8

Collaboration with Department of Pathology, P. Bronsert

31 · April 4, 2019


Characterisation of Extracellular Vesicles by Electron Microscopy

2D

3D

MDA-MB-231 231-Tspan8 MDA-MB-231 231-Tspan8

BT-549

BT-Tspan8

BT-549

BT-Tspan8

MCF7

MCF7-Tspan8

MCF7

MCF7-Tspan8

32 · April 4, 2019


Breast cancer cells produce higher amounts

of small vesicles in 3D than in 2D culture under normoxia and hypoxia

A. MDA-MB-361 B.

MDA-MB-231

231-Tspan8

EVs released per cell

Normoxia

Hypoxia

EVs released per cell

EVs released per cell

EVs released per cell

Normoxia

Hypoxia

C. D.

BT-549

BT-Tspan8

MCF7

MCF7-Tspan8

Normoxia

Hypoxia

Normoxia

Hypoxia

33 · April 4, 2019


Size of EVs produced by the cells, differs under 2D and 3D conditions and

under normoxia and hypoxia

Normoxia

MDA-MB-231

231-Tspan8

BT-549

BT-Tspan8

MDA-MB-361

Hypoxia

MDA-MB-231

231-Tspan8

BT-549

BT-Tspan8

MDA-MB-361

34 · April 4, 2019


To sum up

‣ The 3DCoSeedis TM are applicable for the EV production

‣ 3D conditions differentially affect EV number and size in breast cancer cells

35 · April 4, 2019


Comprehensive analysis of the effect of 3D

condititions on EV release, cargo and function in

gastrointestinal cancer model

Collaboration study between

Medical Center University of Freiburg (Germany) and

Univeristy of Porto (Portugal)

36 · April 4, 2019


Cell spheroids and aggregates grow under controlled conditions in

microwell arrays upto 10 days

Rocha et al,

Advanced Science Feb. 2019

37 · April 4, 2019


Tumor cells produce higher amounts

of small vesicles in 3D than in 2D culture

Rocha et al,

Advanced Science Feb. 2019

38 · April 4, 2019


Overall up-regulation of miRNAs and down-regulation

of proteins in EVs derived from 3D cultures

Rocha et al,

Advanced Science Feb. 2019

39 · April 4, 2019


ARF6 signaling pathway is downregulated in 3D EVs

Rocha et al,

Advanced Science Feb. 2019

40 · April 4, 2019


41 · April 4, 2019

ARF6 signaling pathway is downregulated in 3D EVs


42 · April 4, 2019

ARF6 signaling pathway is downregulated in 3D EVs


Network integrated analysis revealed

a co-regulation of miRNAs and target proteins

Cell

EV

A. Keller, Saabruecken

43 · April 4, 2019


Network integrated analysis revealed

a co-regulation of miRNAs and target proteins

miRNAs

in cells

Target

proteins

in EVs

Rocha et al,

Manuscript in Revision

44 · April 4, 2019


Network integrated analysis revealed

a co-regulation of miRNAs and target proteins

miRNAs

in cells

Target

proteins

in EVs

Rocha et al,

Manuscript in Revision

45 · April 4, 2019


3D conditions modulate functionality of EVs in a cell

line-specific manner

Increased uptake of 3D EVs

Mediation of invasive cell behavior

*

*

46 · April 4, 2019


Conclusions

‣ 3DCoSeedsis TM are applicable for EV isolation, analytic and functional

characterization

‣ Using the 3D microwell array more EVs/cell can be isolated in a considerably more

cost- and efforts- efficient manner than 2D culture

‣ 3D environment is likely to trigger release of smaller EVs with increased amounts of

certain miRNAs and decreased amounts of their target proteins

‣ Functional impact of 3D environment on EVs differs between cell lines and should be

individually analyzed

47 · April 4, 2019


https://www.extracellular-vesicles.de/

48 · April 4, 2019


49 · April 4, 2019

Thery et al., JEV 2018

(ca. 80 co-authors, members of the ISEV community)


International Society of Extracellular Vesicles -

annual meeting ISEV2019 in Kyoto, Japan

German and Austrian Societies of Extracellular Vesicles -

Autumn meeting and HandsOn Workshop in Freising, Munich, Germany

50 · April 4, 2019


Exosomes and Tumor Biology Group

Our Collaborations:

Freiburg:

Andreas Thomsen (Radiology)

Nikolas von Bubnoff (Hemathology

Oncology)

Marie Follo (Core Facility)

National:

Karlsruhe

Christoph Koos (KIT)

Saabruecken

Andreas Keller (Medical Bioinformatics)

Jena

Chirstoph Kraft, Frank Garwe (IPHT);

MSC/ITN Marie Curie

Train-EV

International:

Carla Oliveira, Sara Rocha

(Ipatimub, Porto)

Jean-Charles Sanchez,

Domitille Schvartz (Human Protein

Science, University of Geneve)

apc Biopply (Switzerland)

51 · April 4, 2019

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