Download COST Action BM1204 EU Pancreas Short Term Scientific Missions

COST Action BM1204
EU Pancreas
Short Term Scientific Missions - Final report
“From patient derived pancreatic carcinoma xenografts to
cancer stem cells”
Name of grantee: Deepak Vangala, Ruhr-University Bochum
Date of STSM: 29/09/2013 to 04/10/2013
Host institution: CNIO, Madrid, Stem cells and cancer group, Prof. C.
Heeschen
Grant requested: 766 €
1. Purpose of the STSM
The purpose of the STSM , which took place from September 29th 2013 to October 4th
2013 in the lab of Prof. Christopher Heeschen at the CNIO in Madrid, was to receive
practical training in advanced cell culture techniques regarding the cultivation and
processing of primary pancreatic cancer cells from patient derived xenograft (PDX)
tumors.
The recently described PDE6D-protein (Zimmermann et al, Nature 2013) may be a
new target for the treatment of pancreatic cancer. In order to funtionally further
characterize this protein, we not only want to study inhibtion and overexpression of
PDE6D in established cell lines but also in primary cells from patient derived pancreatic
xenograft tumors. Furthermore, cancer cells with stem cell characteristics are going to
be studied more intensively. This will be conducted not only under regular cell culture
conditions but also in a spheroid-culture model.
Especially the techniques regarding isolation of primary cells from PDX, cultivation of
primary cells, cultivation of spheroids, FACS-analysis of primary cells and stem cells
and cell sorting of stem cells are not yet well established in our lab. The aim of the
STSM was to obtain and improve knowledge and skills regarding these methods.
COST is supported by the
EU RTD Framework
Programme
ESF provides the COST
Office through a European
Commission contract
The Council of the
European Union provides
the COST Secretariat
2. Description of the work carried out during the STSM
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Cell culture conditions for primary cells in adherent and ultra-low adhesion
conditions; composition of media and different additives used for cultivation
-
Sphere forming assays: differences between spheres and aggregates, usage of
cell strainer, Centrifugation conditions
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Sphere counting
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Working with lentivirally transduced spheres
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Microscopy of autofluorescent cells
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Isolation of primary cells from patient derived xenografts, altogether of 4
different tumors
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Strategies for prevention of bacterial and fungal infections
-
Comparison of enzymatic and mechanical dissociation techniques used at the
CNIO and in Bochum
-
Primary cultivation of obtained cells in adherent conditions and in ultra-low
adhesion cell culture flasks
-
Strategies for elimination of Fibroblast outgrowth by FCS-free medium
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Preparation of dissociated cells for FACS analysis
-
Selection of antibody panels for characterizing cancer stem cells
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Strategies for compensation and for data analysis with FlowJo software
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Standardizing sphere forming assays, duration of experiments, time of change
of medium, differences between primary, secondary and tertiary spheres.
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Preparation of primary cells for FACS analysis: Preparation of adherent cells
and preparation of spheres by prolonged trypsinization.
-
Observation of an example of different treatments to one primary pancreatic cell
line in adherent and low adhesion conditions; FACS analysis of both treatment
groups, differential regulation of one target protein
-
Cell sorting: Preparation of Cells, cell counting, incubation with antibodies,
control selection, sorting and collecting buffer, actual sorting
COST is supported by the
EU RTD Framework
Programme
ESF provides the COST
Office through a European
Commission contract
The Council of the
European Union provides
the COST Secretariat
3. Description of the main results obtained
The goal of the STSM namely to obtain and improve knowledge on cell culture
techniques regarding primary pancreatic cancer cells was achieved.
Regarding isolation techniques the question of enzymatic and/ or mechanical
dissociation could be answered, in particular which enzymes to use (collagenase) and
the intensity of mechanical dissociation. A protocol when to use adherent and when to
use low adhesion culture conditions was provided.
One of the main problems in our lab was to separate cell aggregates and spheres into
single cells for FACS-analysis. This problem could be solved by intensive
trypsinization. Besides, a strategy for coping with fibroblast overgrowth in primary
cancer cell cultures was offered by changing medium to serum free medium.
Another important aspect of this STSM was to learn more about FACS-analysis of
primary tumor cells and spheres. Not only were the techniques of cell preparation
(basics such as amount of cells, buffers, incubation time etc.) being taught, but also
was a panel of antibodies for identifying cancer stem cells in pancreatic carcinomas
provided. Furthermore the analysis of the obtained FACS-data to correctly identify
epithelial cells and cancer stem cells was practiced. Finally, one experiment of cell
sorting was conducted.
Besides technical and practical skills, different kinds of experimental problems were
discussed, for example lentiviral transduction protocols and transduction-efficiencies or
the problem of not being able to generate primary cells out of every tumor of one or
different cancer-entities. Besides, the phenomenon of autofluorescence was discussed
extensively. As not all experiments during the STSM worked out successfully (such as
the sell sorting experiments). a wide set of troubleshooting could also be obtained.
In conclusion, the lacking knowledge to successfully continue with the research project
in Bochum could be obtained by the STSM at the CNIO in Madrid.
4. Future collaboration with the host institution
A future cooperation could lay in the exchange of primary tissues for expanding the
number of pancreatic cancer PDX in Bochum and Madrid, respectively.
5. Confirmation by the host institution of the successful execution of the STSM
Added in a separate pdf-file.
COST is supported by the
EU RTD Framework
Programme
ESF provides the COST
Office through a European
Commission contract
The Council of the
European Union provides
the COST Secretariat
6. Other comments
The grantee would like to thank Prof. Heeschen and especially Sonia Alcalá for their
kind hospitality and the well planned training.
COST is supported by the
EU RTD Framework
Programme
ESF provides the COST
Office through a European
Commission contract
The Council of the
European Union provides
the COST Secretariat