In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Podsumowanie

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

Streszczenie

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche. The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines. Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions. Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs. Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions. However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly. CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs. Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques. In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system. This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

Wprowadzenie

Metastasis is a complex multi-step process that remains poorly understood. The E-selectin/selectin ligand axis has been shown to play an important role in tumor metastasis by promoting primary adhesive interactions between the vascular endothelium and cancer cells^1,2. Endothelial (E)-selectin is a transmembrane protein expressed by activated endothelial cells, while different E-selectin ligand(s) are expressed by tumor cells³. Numerous in vitro approaches have been successfully employed to model E-selectin/selectin ligand interactions between tumor cells and endothelial cells (ECs)¹. To study these interactions, different flow chamber systems are being employed to simulate blood vascular system. Among flow chamber assemblies, parallel-plate flow chamber (PPFC) in conjunction with ECs is routinely used as an in vitro model simulating in vivo shear stress conditions. In this method, ECs are grown on a 35-mm dish and after achieving a monolayer, ECs are attached to the PPFC and shear stress based experiments are performed. 

However, PPFC and other current systems present many limitations to studying adhesive interactions between circulating tumor cells (CTCs) derived from patients and ECs, primarily, because CTCs are a rare population of cells, shed from the primary tumor, circulating among millions of blood cells (1 CTC per 10⁹ blood cells)⁴. Hence, unlike unlimited supply of cultured cell lines, low CTC counts lead to very few and rare CTC/EC interactions, requiring proper flow channel width to record the interactions for playback analysis. Additionally, since patient derived CTCs are an impure population, therefore an identification marker is required to track CTCs in specific. To solve this problem, we developed a new method to identify prostate cancer (PCa) CTCs by taking advantage of the fact that virtually all of these CTCs express prostate specific membrane antigen (PSMA) on their cell surface^5,6. In this report, we used the prostate cancer cell line, MDA PCa2b (MDA), to demonstrate the potential utility of our new system to study prostate CTC interactions with ECs, eventually to understand the mechanism of metastasis.

Our methodology can be applied for various shear based experiments simulating in vivo vascular system^7-9. Besides examining PCa CTC/EC interactions, the current flow chamber system could be easily adapted for analyzing peripheral blood mononuclear cells or tumor cells’ interactions with ECs. The ease of disassembling and reassembling of the flow chamber, a microslide III ^(0.1) (hereafter referred as microslide), allows culturing ECs under perfusion and stimulating ECs with different cytokines to induce protein expression. Besides, cultured ECs, recombinant proteins such as E- and P-selectin can be coated onto the microslide and interactions with tumor cells can be observed under laminar flow conditions¹⁰.

Protokół

1. Culturing HUVECs on Microslides for Observing CTC-endothelial Interactions

1. Under the tissue culture hood, first rinse the microslide, channel width of 1 mm with PBS. Gently coat the microslide with 200 µl of 50 µg/ml fibronectin (dissolved in PBS) using a 1-ml Luer-lock syringe.
2. Cover the microslide with the lid and keep it inside the tissue culture hood for 30 min. Slow dispensing of the liquid in the microslide prevents bubble formation in the channel.
3. Perfuse 200 µl of warm (37 °C) HUVEC growth medium (M199 media, 1M HEPES, 20% FBS, 5 mg/ml heparin, 100 µg/ml endothelial cell growth factor, and L-glutamine) over the microslide and incubate for 20 min at RT. During the perfusion, prepare HUVEC cell suspension.
4. Rinse HUVECs with PBS and add 0.05% trypsin-EDTA for 1-2 min at RT. Centrifuge HUVECs in 2 ml growth medium at 180 x g for 5 min.
5. Measure the cell concentration using a neubauer hemocytometer and prepare 10⁷ HUVEC cells/100 µl growth medium. Then, carefully remove the medium from the inlet of the microslide using a 200-µl pipette tip.
6. Bring the microslide to eye level and using a 1-ml Luer-lock syringe, gently perfuse 200 µl of prepared concentration of HUVECs into the channel. Caution is required at this step to prevent bubble formation. If the bubbles appear, keep perfusing for a slightly longer time until bubbles enter the outlet channel.
7. Put an equal volume (~80 µl) of HUVEC media in both the inlet and outlet of the microslide. This prevents the flow of cells in either direction.
8. Cover the slide and keep it in the incubator (37 °C) for 1.5 hr.

2. Preparation of Flow Chamber Assembly for Overnight HUVEC Culture on a Microslide

1. Place a sterile 20-ml syringe, female and male Luer connectors, tubing, and a syringe pump in the incubator for 15 min.
2. For minimal dead volume, use the tubing with inner diameter of 0.04 inches. Smaller tubing diameter prevents bubble formation.
3. Fill the 20-ml syringe with warm (37 °C) HUVEC media (12 ml). Attach the tubing with the connectors onto the syringe. Remove the bubbles. Connect this assembly to the microslide.
4. Completely fill the inlet of the microslide with HUVEC media. Bring the filled 20-ml syringe attached to the connector next to the microslide. Gently attach the connector to the microslide.
5. Bring the set-up into the incubator and connect it to the syringe pump, set at 10 µl/min shear rate. Leave the cells O/N in the incubator at 37 °C.
6. Next day, disassemble the set-up by removing the connector attached to the inlet of the microslide.
7. To upregulate E-selectin expression on ECs, prepare fresh growth medium containing IL-1β at 10 ng/ml in 4 ml media. Aspirate the media in a 10-ml syringe. Remove the media from the inlet of the microslide and connect the syringe to the slide.
8. Set the syringe pump at 10 µl/min shear rate for 4 hr in the incubator.

3. Preparation of Anti-PSMA (J591-488) Labeled Prostate Cancer Cells

1. During IL-1β incubation of HUVECs, prepare anti-PSMA J591-alexa488 labeled PCa cells. Add 0.05% trypsin-EDTA to MDA cells for 1 min. Do not expose the cells to trypsin for longer times as it can affect the glycopeptides present on the cell surface. Enzyme free cell dissociation reagent can also be used instead of trypsin. Centrifuge at 200 x g for 5 min.
2. Resuspend MDA cell pellet in 1 ml H/H buffer (Hanks balanced salt solution/0.1% HSA/10 mM HEPES/1 mM CaCl₂). Add anti-PSMA J591-alexa488 antibody at 20 µg/ml for 30 min at RT in a dark place. Resuspend the cells during the incubation.
3. After 30 min, centrifuge the cell solution at 800 x g for 5 min. Aspirate and resuspend the pellet in 1 ml H/H buffer. Count the labeled MDA cells and bring the final concentration to 1x10⁶ cells/ml. Fill a 5-ml syringe with J591-488 labeled MDA cells and remove the bubbles.

4. Preparation of Anti-PSMA (J591-488) Labeled CTCs Enriched from PCa Patients

1. Collect 7.5 ml blood from prostate cancer patients in a blue cap tube (containing sodium citrate). Gently dilute blood 1:1 in 0.1% BSA/1 mM EDTA/PBS.
2. Add 5.3 ml Ficoll-paque plus in a 50-ml conical tube. Layer diluted blood on top of the ficoll-paque. Centrifuge at 400 x g for 30 min.
3. Pre-coat all the tubes or tips coming in contact with CTCs with 2% FBS/RPMI-1640/ 1 mM CaCl₂/ 4 mM MgCl₂ (R/S buffer) to prevent non-specific binding of CTCs to the surfaces. Maintain 4 °C temperature of media and buffers coming in contact with CTCs.
4. Collect the peripheral blood mononuclear cell (PBMCs) fraction containing CTCs from the interface in another 50-ml conical tube containing 30 ml R/S buffer. Centrifuge at 400 x g for 8 min.
5. Resuspend the pellet and wash again in R/S buffer at 400 x g for 8 min. After centrifugation, resuspend the pellet in H/H buffer. Add anti-PSMA J591-488 antibody at 20 µg/ml for 30 min at RT in a dark place.
6. After 30 min, centrifuge the PBMCs containing J591-488 labeled CTCs at 800 x g for 5 min. Resuspend in H/H buffer. Fill a 1-ml syringe (pre-coated with R/S buffer) with the sample.

5. Preparation of Microscope, Syringe Pump and Flow Chamber Assembly

1. Turn on the inverted microscope and set the Kohler illumination at 10X objective. Bring the syringe pump at the same level as the sample stage of the microscope and set it at 1 dyn/cm² shear stress (~ 10 µl/min).
2. Open the Zeiss Axiovision software. Select the objective on the computer screen. Create a new folder under the tools option in the software.
3. Open the smart experiments option in Axiovision and adjust the settings to record short 30 sec videos for 30 min.
4. For live fluorescent video, set the image options- 12 msec Exposure, 2 x 2 bin, 5 Gain. These parameters help in attaining videos close to the video frame rate (~ 23 frames per sec) with the Zeiss mRm camera.
5. To visualize full width of the flow channel at 10 X objective on the computer screen, use a C-mount adapter (0.63 x f/60 mm Interface).
6. Switch on the mercury lamp.
7. Place the syringe and the connector containing the cells onto the syringe pump.
8. Bring IL-1β stimulated HUVECs from the incubator. Attach the connector to the filled inlet channel of the microslide containing HUVECs.
9. Connect the outlet channel with the connector attached to the tubing. Put the tubing into a dish or 15 ml conical tube to collect the flow through.
10. Start the infusion through the microslide at 10 µl/min. Observe the interactions between endothelial cells and labeled MDA cells or labeled CTCs derived from patients under 488-nm filter on the epifluorescence microscope.
11. Start recording the experiment as 30 sec short videos. During playback analysis, measure the rolling velocity. Rolling velocity is measured by dividing the distance traveled by the cells over time.

6. Immunostaining of the Microslide

1. Remove the media from the inlet and outlet of the microslide after perfusing MDA cells on IL-1β-stimulated HUVECs for 10 min.
2. Using a 200-µl tip, put warm (37 °C) PBS containing calcium and magnesium into the inlet of the microslide for 5 min. Tilt the microslide using its lid as a prop on the bench. Keep the microslide in a tilted position for all the incubations during immunostaining.
3. Fix HUVECs by perfusing warm 2% formaldehyde into the inlet
4. Incubate for 20 min at RT. Rinse twice with PBS for 5 min each.
5. Add triton-X 100 (0.1%) in 5% BSA in PBS for 10 min at RT.
6. Rinse twice with PBS for 5 min each. Block with 5% BSA in PBS for 30 min.
7. Incubate with primary antibody goat anti-VE-Cadherin (1:100) in 2.5% BSA O/N at 4 °C.
8. Next day, wash twice with PBS for 5 min each. Add secondary donkey anti-goat 647 antibody for 45 min. Wash twice with PBS for 5 min each.
9. Incubate at RT with humanized J591-alexa488 conjugated antibody for 1 hr.
10. Wash twice with PBS for 5 min each. Add DAPI for 5 min. Wash with distilled water for 5 min.
11. Add PBS (or mowiol for long-term storage). Visualize the microslide under the confocal microscope.

Wyniki

Figure 1 shows an O/N culture of a monolayer of ECs on the microslide. The scalings on Figure 1A shows that 100% of the microslide is visible using 5X objective while 70% is visible using a 10X objective (Figure 1B). For E-selectin mediated interactions, cells rolling at the edges are not considered which makes more than 70% of the microslide available for video-recording and playback analysis. In our experience, initially this set-up assembly needs some practice to cult...

Dyskusje

Due to the low number of CTCs among blood cells, it is difficult to isolate CTCs as a pure population of cells. In order to study CTC/EC interactions, the rare and impure population of CTCs poses two major challenges: a) Identification of CTCs among blood cells; b) Observation of CTC/EC interactions.

To overcome the first limitation of identifying prostate CTCs among blood cells, we took advantage of the fact that virtually all prostate tumor cells express PSM...

Materiały

Name	Company	Catalog Number	Comments
Microslide	Ibidi	80331
Fibronectin	Millipore	FC010
Plastic tubing	Cole Parmer	EW-96115-08
Male Luer adapter	GlycoTech	31-001
Female Luer adapter	GlycoTech	31-001
Syringe pump	Chemyx Inc	Fusion 100
Luer-lock syringe	BD Biosciences	309628
M199 medium	Sigma	M7653
Endothelial Mitogen	Biomedical Technologies	BT-203
HBSS	Sigma	H9269
Anti-PSMA J591-488	Weill Cornell Medical College-Lab of Urologic Oncology
Interleukin-1 beta	Peprotech	200-01B
Trypsin	Millipore	SM-2002-C
Heparin	Sigma	H-3149
HUVECs	Weill Cornell Medical College-Department of Surgery		provided by Marco Seandel
VE-Cadherin	Santa Cruz	sc-5648
10x objective	Zeiss Plan Neofluar
Enzyme free cell dissociation reagent	Millipore	S-004-C
RPMI-1640	Lonza	12-702-F
Ficoll-paque plus	GE healthcare	17-1440-02