On the search for double positives
Do cells with a CK+/VIM+ phenotype exist in
colorectal cancer and are these cells a
morphological expression of EMT?
Sara Meyer, medical student (5th year)
Institute of Pathology
University of Berne, Switzerland
26.9.2016 Congress of the ESP, Cologne
Background
What is tumor budding?
100 𝜇𝑚
CK Staining, 20x, CRC Invasion front
2
Background
What is tumor budding?
>
What are tumor buds?
— Tumor cell clusters up to five cells at the invasion front
— Loss of adhesion molecules such as E-Cadherin
>
Budding markers
— Loss of a stable cell-cell adhesion
— Tumor buds take up CK-staining (epithelial marker)
>
Tumor budding as a prognostic indicator
>
 Morphological expression of EMT?
3
Background
What is EMT?
Epithelial Mesenchymal Transition
Double positive
phenotype
CK+ VIM+
Picture: Kalluri R, Weinberg R, The basics of epithelial-mesenchymal transition (2009) in: J. Clin. Invest, p.1421
„Hybrid state“: Jolly MK, Implications of the hybrid epithelial/mesenchymal phenotype in metastasis (2015) in: Front. Oncol
4
Hypothesis & Aim
A small subgroup of tumor buds will have a double positive
phenotype, because they are transitioning from an epithelial
to a mesenchymal state.
 Identify double positive cells in colorectal cancer
 Determine their origin
5
Methods
Workflow Overview
Exploration of
single cells
Goal:
Find
double
positives
High
grade
budding
cases
• Annotations and
Punching
• DEPArray
• Sequencing and
Quantification
Confocal microscopy
• Immunostaining and
fluorescence labeling VIIM
& CK
6
Methods
Exploration of single cells
>
Immunostaining with PanCK and scanning
>
Annotations
>
Punching
>
DEPArray
— Disaggregation into single cells
— Fluorescent staining with CK and VIM
— Separated by Dielectrophoresis-Array
>
Sequencing of recovered cells
7
Results
Exploration of single cells
CK staining, 10x, CRC sample 1
8
Results
Exploration of single cells
VIM+ / CK-
✕
✔
✔
VIM- / CK+
✔
✔
✕
VIM+ / CK+
✕
✔ 3%
✕
BRAF mutation
✔
✔
✕
TP53 mutation
✔
✔
✕
PTEN deletion
✔
✔
✕
9
Results
Exploration of single cells
VIM+ / CK-
✕
✔
✔
VIM- / CK+
✔
✔
✕
VIM+ / CK+
✕
✔ 3%
✕
BRAF mutation
✔
✔
✕
TP53 mutation
✔
✔
✕
PTEN deletion
✔
✔
✕
10
Methods
Confocal microscopy
>
Searching CK stainings with regions of high concentrated
tumor buds
>
Immunostaining with VIM, CK and E-Cad
>
Immunofluorescence
— DNA marker: DAPI
— mesenchymal marker: VIM
— epithelial marker: CK or E-Cadherin
>
Searching for double positives
11
Results
Confocal microscopy
50 𝜇m
CK-VIM-DAPI 60x
12
Results
Confocal microscopy
50 𝜇m
Ecad-VIM-DAPI, 60x
50 𝜇m
Ecad-DAPI, 60x
Results
Confocal microscopy
>
Supporting the search of double positives with a different
sample
>
TMA of densest vessles buds
100 𝜇𝑚
TMA, H&E staining, 10x
CRC, D2-40/CK doublestaining with annotations, 10x
14
Results
Confocal microscopy
50 𝜇m
CK-VIM-DAPI, 60x
Courtesy of José Galvan, TRU
15
Discussion
>
3% of detected tumor buds have a double positive phenotype
>
Originating from tumor
>
Partial EMT
>
EMT important for the understandig of metastasis and for
providing an adequate therapy
16
Acknowledgements
>
Prof. Dr. Inti Zlobec
>
Dr. Irene Centeno-Ramos & Dr. José Galván
>
Dr. Lena Sokol & Stefan Zahnd
>
Liliane Schöni & Patricia Ney
>
Dr. med. Heather Dawson
>
Prof. Dr. med. Alessandro Lugli & Prof. Dr. med. Aurel Perren
>
And all the other members of the CRC- and GI-group
17
Extra Slides
18
Sources
>
Jolly MK, Implications of the hybrid epithelial/mesenchymal phenotype in metastasis
(2015) in: Front. Oncol.
>
Galvan J, Zlobec I, Lugli A, ... TWIST1 and TWIST2 promoter methylation and protein
expression in tumor stroma influence the epithelial-mesenchymal transition-like tumor
budding phenotype in colorectal cancer (2014) in: Oncotarget
>
Koelzer V, Zlobec I, Berger M, Dawson H, Lugli A, ... Tumor budding in colorectal cancer
revisited: results of a multicenter interobserver study (2015) in: Virchows Archiv
>
Kalluri R, Weinberg R, The basics of epithelial-mesenchymal transition (2009) in: J. Clin.
Invest
>
Silicon Biosystems S.p.A. DEPArray Technology brochure, from:
http://www.siliconbiosystems.com/deparray-system
19
Outlook
>
Fluorescence activated cell sorting FACS
— deparaffinization & dissociation into single cells
— antibody labelling: CK, VIM, DAPI
— detecting VIM/CK-double positives ～3%
20
Methods
DEPArray
>
Dielectrophoresis (DEP) + Array
>
image-based cell selection to identify and recover specific
individual cells of interest from complex, heterogeneous
samples
21
>
>
>
>
>
>
>
Staining with Keratin-Alexa 488 (green)
Vimentin-Alexa 647 (red)
DAPI
Ion Torrent 50 gene panel sequencing
(found: BRAF, PTEN, TP53)
Next step: TMA analyses of double positives
Next step: in-house single cell sorting through FACS
Next step: Nanostring for mRNA-Expression of gene panel
— Characterize recovered double positive cells using Nano-string
for EMT related genes
(also: compare CK+/Vim- Tumor cells and Buds to the double
positives Buds)
>
Next step: Laser capture tumor buds
22
NGS
>
Next generation sequencing (NGS, NextGenSeq) is a new
method for sequencing genomes at high speed and at low
cost. It is also known as second generation sequencing
(SGS) or massively parallel sequencing (MPS).
23
Nanostring
>
>
The NanoString nCounter Analysis System delivers direct,
multiplexed measurement of gene expression, providing
digital readouts of the relative abundance of hundreds of
mRNA transcripts simultaneously.
nCounter System delivers accurate gene expression data
directly from crude FFPE extracts, without requiring RNA
purification
24
Cancer Panel
>
>
The Ion AmpliSeq Cancer Hotspot Panel v2 allows
translational and disease researchers to fast-track oncology
research by surveying hotspot regions of 50 oncogenes and
tumor suppressor genes, with wide coverage of the KRAS,
BRAF and EGFR genes.
This research panel, with improved primer design, contains
207 primer pairs in a single tube and requires as little as 10ng
of DNA, enabling researchers to sequence challenging
samples such as formalin-fixed, paraffin-embedded (FFPE)
tissue.
25
Cancer Panel
26
Cancer Panel
27
CK (AE1/AE3) Staining, CRC, 10x
BRAF (V600E) Staining, CRC, 10x
29
BRAF (V600E) Staining, Tumor Buds, 40x
30
Results
Sample Number 3 (49 V+K+)
31