2 0 0 0 0 isolation of osteosarcoma circulating …...parsortix®: a microfluidic device for...
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Dominique Heymann & Robin YoungUniversity of Sheffield
2000029.11
7.12
Isolation of osteosarcoma circulating tumour cellsusing microfluidic devices
• Osteosarcoma patients with metastatic disease have a poor prognosis
• The study of CTCs provide a window into the metastatic process– Understanding tumour biology, identifying novel therapeutic targets
• Enumerating CTCs may be clinical relevant – Potential prognostic marker, to stratify treatments, surveillance to detect
recurrence earlier
Circulating tumour cells (CTCs)
(Tellez-Gabriel et al Front Oncol 2016)
(Zhang et al Int J Oncol 2017)
Method: Aneuploidy, assessed by flurorescence in situ hybridisation,
and CK18-/CD45- phenotype
Detection of CTCs in osteosarcoma patients
(Wu et al Cancer Manag Res 2018)
Enneking stage
IIB 19 patients
III 9 patients
Method: Enrichment by CanPatrolTM = filtration system, then CTC
characterisation by RNA-in situ hybridisation (Vimentin, Twist)
Detection of CTCs in osteosarcoma patients
Depletion of leukocytes and
residual thrombocytes
Anti-TERT119
+ anti-CD45
Reduction of
blood volume
Centrifugation
Red cell lysis
(2 x)
Depletion of residual
and collect of
enriched cancer cells
Anti- CD45 APC
+
GFP-cancer cells
Separation
column
Time course
Cell culture + Single cell
analysis (RTqPCR)
DEPArrayTM
cell sorting
Paratibial inoculation of
GFP-MNNG/HOS cells
Flow cytometry
(Chalopin et al J Bone Oncol 2018)
CTCs: biomarker for clinical follow up?
Method
D0 D45
1.5x106
GFP MNNG/HOS Sacrifice
n=10
D10 D30
Early detection of CTCs in blood
****
No detectable CTCs at 24h and 48h after inoculation of cancer cells
(Chalopin et al J Bone Oncol 2018)
Detecting CTCs: a technical challenge
➢ 50 different CTC assays
➢ Technical challenge:
detection and isolation
of one CTC amongst
106-108 normal blood
cells
(Alix-Panabieres and Pantel Clin Chem 2012)
Pre-requisite: ENRICHMENT METHODS
Parsortix®: a microfluidic device
for enriching circulating tumour cells
Flow
Flow
Flow
1)Bloodsample 2)EnrichmentusingParsor x3)Collec onofenriched
sample4)Isola on,quan fica onand
characteriza onofCTCsusingDEPArray
HKBC00001a-P22W10R0017-20170207-1HKB7C
Group brightfield_0 fitcsb_1 brightfield_fitcsb_2
cell_id = 134row_num = 1
Hi GFP
cell_id = 125row_num = 2
Hi GFP
cell_id = 121row_num = 3
Hi GFP
cell_id = 120row_num = 4
Hi GFP
cell_id = 114row_num = 5
Hi GFP
cell_id = 112row_num = 6
Hi GFP
www.siliconbiosystems.comPage 1
Blood sample: 10 mL (Streck tube)
DEPArray™ NxT Instrument: Image-based digital sorter
DEPArray™ A300K: semiconductor cartridge
+
Single cell isolation for molecular characterisation
Method: DEPArrayTM
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nDEP Cage nDEP Cage
+ +++ --
Non-uniform electric field
generated by the chip electrodes (cross section)
Cell trapping by DEP cages
Cage-move
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Single cell isolation for molecular characterisation
Method: DEPArrayTM
Single cell isolation for molecular characterisation
Method: DEPArrayTM
aneuploid
(unpublished data)
- Osteosarcoma patient (Newcastle, Mr Rankin): 25 years old, proximal humerus,
recurrent disease with lung metastases after two years
- Methods: 10 mL of blood
(unpublished data)
Flow
Flow
Flow
1)Bloodsample 2)EnrichmentusingParsor x3)Collec onofenriched
sample4)Isola on,quan fica onand
characteriza onofCTCsusingDEPArray
HKBC00001a-P22W10R0017-20170207-1HKB7C
Group brightfield_0 fitcsb_1 brightfield_fitcsb_2
cell_id = 134row_num = 1
Hi GFP
cell_id = 125row_num = 2
Hi GFP
cell_id = 121row_num = 3
Hi GFP
cell_id = 120row_num = 4
Hi GFP
cell_id = 114row_num = 5
Hi GFP
cell_id = 112row_num = 6
Hi GFP
www.siliconbiosystems.comPage 1
Isolation of Osteosarcoma Cells by
DEPArrayTM: proof of concept
(unpublished data)
Isolation of CTCs in Osteosarcoma Patient
. 2.8 cells/mL whole blood (Vimentin+ cells with abnormal DNA content)
. 10.5 cells/mL whole blood (Vimentin+Keratin+ cells with abnormal DNA content)
Questions?