CTC Isolation from a Whole Blood Sample: A Method to Obtain CTCs from Murine Colorectal Cancer Model

Protocol

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Isolation of Circulating Tumor Cells from Whole Blood Samples

NOTE: Obtain blood by transcutaneous cardiac puncture on anesthetized mice, followed by euthanasia. Ideally, 1,000 µL of blood are drawn into a syringe prefilled with 100 µL of an anticoagulant (Ethylenediaminetetraacetic acid (EDTA) or heparin). Use an anti-human-EpCAM (epithelial cell adhesion molecule) antibody to identify the CTCs. This works very well if human epithelial cell lines are used in the mouse model. For other applications, different antibodies may be required.

1. CTC enrichment:
   1. Prefill 15 mL tubes with 5 mL density gradient medium and carefully transfer the blood into the 15 mL tube.
   2. Centrifuge (30 min at 300 x g without brake) and carefully remove the upper supernatant.
   3. Pour the rest into a new 15 mL tube and centrifuge (15 min, 300 x g with brake).
   4. Recover the interphase containing the mononuclear cells (discard the rest) and wash the cells twice with PBS.
   5. Resuspend the pellet in 200 µL PBS/1% EDTA and add 4 µL of EpCAM antibody. Incubate 20 min on ice in the dark.
2. Screening and picking
   1. Prepare the following buffer (in the following referred to as picking buffer): PBS + 10% fetal calf serum (FCS) + 1% Penicillin/Streptomycin (1%) + 0.8% EDTA.
   2. Use a PAP pen to draw a ~1 cm circle in a 6 cm sterile Petri dish (prevents the fluid from dispersing in the dish), and pipet 700 µL of picking buffer into this circle.
   3. Add 50 µL of cell suspension to the 700 µL picking buffer within the circle. Check the density of cells with the microscope. Split the sample into different dishes if it is too dense.
   4. Wait for the cells to settle down (~5 min).
   5. Screen the drop of cell suspension for stained (= EpCAM-positive) cells.
   6. Once a cell is found, pick the cell with the micromanipulator and put it in a 50 µL buffer depending on the intended downstream analysis (e.g., RNA extraction buffer or culturing medium).
   7. Depending on the intended downstream analyses, isolate EpCAM-negative cells and/or medium as negative controls.
   8. Proceed with downstream analyses (e.g., cell culture, PCR).

Materials

Name	Company	Catalog Number	Comments
Density gradient medium – Ficoll	StemCell - Lymphoprep	7801
Alexa Fluor 488 anti-human CD326 (EpCAM) Antibody clone 9C4	BioLegend	324210
Alexa Fluor 488 anti-mouse CD326 (EpCAM) Antibody clone G8.8	BioLegend	118210
Petri Dish, 60 x 15 mm, 21 cm², Vent	Greiner bio-one	628102
Fluorescence Cell Culture Microscope	Leica
Transferman 4r Micromanipulator	Eppendorf
CellTram Air	Eppendorf		aspiration pump connected to the micromanipulator
Dmz Universal Microelectrode Puller	Dagan Corporation		required for the manufacturing of microcapillaries for single-cell aspiration
Prism Glass Capillaries	Dagan Corporation
PAP pen	Abcam
Dulbecco's Phosphate Buffered Saline	Life Technologies GmbH	14190169	picking buffer
Fetal Calf Serum (FCS)	BIOCHROM AG	S 0115	picking buffer
Penicillin/Streptomycin (PenStrep)	Life Technologies GmbH	15140122	picking buffer
EDTA	Roth	8040.1	picking buffer
Purified anti-human CD326 (EpCAM) antibody clone 9C4	BioLegend	324201	EpCAM immunohistochemistry
HRP rabbit anti-mouse IgG	Abcam	ab97046	EpCAM immunohistochemistry