Establishment and evaluaon of a novel method ... - ANGLE plc...We would like to thank Angle PLC for...
1
Acknowledgements We would like to thank Angle PLC for giving us the opportunity to develop this protocol by using the Parsorx system. Special thanks goes to Sabine Hofmeister and the team of the immunocytology laboratory of the University Women’s Clinic in Tübingen and to all paents for donang bone marrow. 7 Summary - Spiked tumor cells in bone marrow can be isolated by Parsorx - Newly established protocol increases the amount of recovered cells by 60% Outlook -> 1 st experiments for isolaon of paent DTCs look promising -> Establish workflow for downstream analyses of captured DTCs 6 Results III + 5. Run in Parsorx 1. Filter (100 µm) 3. Centrifuge 2. Wash 4. Resuspend pellet Count ‘green’ cells (microscope) 0 20 40 60 80 100 % Capture of spiked cells (’green’) 0 20 40 60 80 100 % Recovery of spiked cells from cassette Establishment of a protocol for processing paent bone marrow before running through Parsorx -> The newly established protocol allows a recovery of about 80% of input target cells -> 75% of captured cells can be harvested for downstream analyses -> Low pressure run drascally reduces the amount of cells during harvesng process 5 Results II Establishment of a new work protocol without PBMC-isolaon needed Bone marrow Filter (100 µm) + X Run in Parsorx -> Running filtered bone marrow clogs the cassees 4 Results I Standard PBMC-isolaon results in an 80% target cell loss 0 20 40 60 80 100 % Recovery of spiked cells (’green’) Bone marrow Ficoll PBMC Pellet Centrifugaon SK-BR-3 cells (CellTrackerGreen) Cytospin (PBMC) Count ‘green’ cells (microscope) 0 20 40 60 80 100 % Capture of spiked cells (’green’) Parsorx (PBMC) -> Parsorx seems to capture a high amount of available cells in the PBMC-fracon 3 Parsorx Cross secon through cassee Principle of marker-independent enrichment of tumor cells by a microfluidic plaorm Plan view Captured CTC Blood flow White blood cell Red blood cell Tumor cells? - Size-based capture of tumor cells - Blood cells can pass through - Various step sizes available (6/10 µm) - Automated in-cassee anbody staining - Harvest of cells out of the cassee for downstream analysis Example of captured CTCs 2 Iliac crest Standard DTC-detecon Bone marrow aspiraon Immunohistochemistry Analysis Standard procedure of DTC-detecon in bone marrow form cancer paents Bone marrow Ficoll PBMC Pellet Centrifugaon PBMC isolaon ≈20% of cancer paents are DTC-posive (detected by Pan-Cytokeran-anbody) 1 ABSTRACT Background: The disseminaon of individual tumor cells is a common phenomenon in solid epithelial cancers. In primary breast cancer these disseminated tumor cells (DTCs) have been found in the bone marrow (BM) of around 20-30% of the paents. The detecon of DTCs has been correlated not only to metastac relapse but also to locoregional relapse. Characterizing DTCs is oſten difficult due to their general rarity in BM aspirates. The method of choice for detecng DTCs is an anbody-based staining for various cytokerans aſter density gradient centrifugaon of the BM to enrich for mononuclear cells. In experiments with spiked cancer cells we show that this procedure is accompanied by a tremendous reducon of target cells with an average of up to 80% cell loss. Methods: Here we provide a fast and easy method for bone marrow preparaon to enable the use of the size based cell enrichment system, Parsorx TM . The system has already been established as a tool for capturing circulang tumor cells (CTCs) from cancer paent blood. The here established and evaluated method helps to increase capture rate of inially spiked cancer cells in paent bone marrow to 75% on average. Results and Conclusion: The Parsorx TM microfluidic plaorm has recently been established for the size based enrichment of CTCs from cancer paent blood. This is the first report on a successful establishment of a protocol for detecng DTCs using cancer paent bone marrow with this device. The method is easy to use and can presumably be applied to other body fluids for detecon of tumor cells in liquid biopsies. Establishment and evaluaon of a novel method for size separaon of disseminated tumor cells from cancer paent bone marrow by a microfluidic plaorm ANDRÉ KOCH, FLORIN-ANDREI TARAN, SARA Y. BRUCKER, ANDREAS D. HARTKOPF Department of Women’s Health, Universityclinic Tuebingen, Calwerstrasse 7, 72076 , Tuebingen, Germany 3 rd ACTC Advances in Circulang Tumour Cells Liquid Biopsy in Clinical Pracce October 4th - 7th, 2017, Rhodes, Greece Department für Frauengesundheit
Transcript of Establishment and evaluaon of a novel method ... - ANGLE plc...We would like to thank Angle PLC for...
AcknowledgementsWe would like to thank Angle PLC for giving us the opportunity to develop this protocol by using the Parsortix system. Special thanks goes to Sabine Hofmeister and the team of the immunocytology laboratory of the University Women’s Clinic in Tübingen and to all patients for donating bone marrow.
7 Summary- Spiked tumor cells in bone marrow can be isolated by Parsortix
- Newly established protocol increases the amount of recovered cells by 60%
Outlook-> 1st experiments for isolation of patient DTCs look promising
-> Establish workflow for downstream analyses of captured DTCs
6 Results III
+
5. Run in Parsortix
1. Filter (100 µm)
3. Centrifuge
2. Wash 4. Resuspend pelletCount ‘green’ cells
(microscope)
0
20
40
60
80
100
% C
aptu
re o
f spi
ked
cells
(’gr
een’
)
0
20
40
60
80
100
% R
ecov
ery
of s
pike
d ce
lls fr
om c
asse
tte
Establishment of a protocol for processing patient bone marrow
before running through Parsortix
-> The newly established protocol allows a recovery of about 80% of input target cells
-> 75% of captured cells can be harvested for downstream analyses
-> Low pressure run drastically reduces the amount of cells during harvesting process
5 Results IIEstablishment of a new work protocol without PBMC-isolation needed
Bonemarrow
Filter (100 µm)
+ XRun in Parsortix-> Running filtered bone marrow
clogs the cassettes
4 Results IStandard PBMC-isolation results in an 80% target cell loss
0
20
40
60
80
100
% R
ecov
ery
of s
pike
d ce
lls (’
gree
n’)
Bonemarrow
Ficoll
PBMC
Pellet
Centrifugation
SK-BR-3 cells(CellTrackerGreen)
Cytospin(PBMC)
Count ‘green’ cells(microscope)
0
20
40
60
80
100
% C
aptu
re o
f spi
ked
cells
(’gr
een’
)
Parsortix(PBMC)
-> Parsortix seems to capture a high amount of available cells in the PBMC-fraction
3 Parsortix
Cross section through cassette
Principle of marker-independent enrichment of tumor cells by a microfluidic platform
Plan view
Captured CTC
Blood flow
White blood cell
Red blood cell Tumor cells?
- Size-based capture of tumor cells
- Blood cells can pass through
- Various step sizes available (6/10 µm)
- Automated in-cassette antibody staining
- Harvest of cells out of the cassette for downstream analysis
Example of captured CTCs
2
Iliac crest
Standard DTC-detection
Bone marrowaspiration
Immunohistochemistry Analysis
Standard procedure of DTC-detection in bone marrow form cancer patients
Bonemarrow
Ficoll
PBMC
Pellet
Centrifugation
PBMC isolation
≈20% of cancer patients are DTC-positive (detected by Pan-Cytokeratin-antibody)
1 ABSTRACTBackground: The dissemination of individual tumor cells is a common phenomenon in solid epithelial cancers. In primary breast cancer these disseminated tumor cells (DTCs) have been found in the bone marrow (BM) of around 20-30% of the patients. The detection of DTCs has been correlated not only to metastatic relapse but also to locoregional relapse. Characterizing DTCs is often difficult due to their general rarity in BM aspirates. The method of choice for detecting DTCs is an antibody-based staining for various cytokeratins after density gradient centrifugation of the BM to enrich for mononuclear cells. In experiments with spiked cancer cells we show that this procedure is accompanied by a tremendous reduction of target cells with an average of up to 80% cell loss. Methods: Here we provide a fast and easy method for bone marrow preparation to enable the use of the size based cell enrichment system, ParsortixTM. The system has already been established as a tool for capturing circulating tumor cells (CTCs) from cancer patient blood. The here established and evaluated method helps to increase capture rate of initially spiked cancer cells in patient bone marrow to 75% on average. Results and Conclusion: The ParsortixTM microfluidic platform has recently been established for the size based enrichment of CTCs from cancer patient blood. This is the first report on a successful establishment of a protocol for detecting DTCs using cancer patient bone marrow with this device. The method is easy to use and can presumably be applied to other body fluids for detection of tumor cells in liquid biopsies.
Establishment and evaluation of a novel method for size separation of disseminated tumor cells from cancer patient bone marrow by a microfluidic platformANDRÉ KOCH, FLORIN-ANDREI TARAN, SARA Y. BRUCKER, ANDREAS D. HARTKOPF Department of Women’s Health, Universityclinic Tuebingen, Calwerstrasse 7, 72076 , Tuebingen, Germany
3rd ACTC Advances in Circulating Tumour CellsLiquid Biopsy in Clinical Practice
October 4th - 7th, 2017, Rhodes, Greece
Department fürFrauengesundheit
