Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Podsumowanie

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Streszczenie

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5^cre:ZsGreen^l/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

Wprowadzenie

Endothelial cells (EC) are essential during the development of solid tumors. From initiation of the angiogenic switch in dormant tumors to dissemination and seeding of metastases at distant sites, EC form the conduits that provide blood, oxygen, and nutrients to sustain tumor growth ¹. As recently suggested, EC also have perfusion-independent functions and form a niche that supports the growth of cancer stem cells and other tumor stromal cells ^2-5. Thus, highly purified tumor-specific EC (TEC) for in vitro culture allows for routine functional studies that will shed light on novel molecular mechanisms mediating tumor angiogenesis and cross talk with tumor cells.

EC are highly specialized depending on the tissue of origin ⁶. Due to the heterogeneous nature of different tumor types and the tumor microenvironment, TEC may also display unique features that reflect a tumor-specific specialization of the vasculature. For example, there is striking variability in the gene expression signatures in TEC isolated from different types or grades of tumors ^7,8. However, frequent co-purification of non-EC, especially tumor-associated fibroblasts and tumor cells, with TEC can confound genome-wide expression analyses. These unwanted cell types are especially problematic in studies that rely on long-term in vitro expansion of TEC cultures.

Described here is a high-fidelity method that consistently produces pure EC cultures from tumors and other tissues. Following immunomagnetic column enrichment of EC fractions and removal of co-purified non-EC, an additional cloning-ring step to capture pure EC colonies is used ⁹. Each colony can be expanded in culture for multiple passages without the emergence of contaminating non-EC. This method also yields multiple EC clones from a single isolation procedure, which is ideal for the study of endothelial heterogeneity. In addition, it is shown that Cdh5^cre:ZsGreen^l/s/l reporter mice are a valuable tool for generating “fate-mapped” and indelibly-marked EC which maintain ZsGreen fluorescence in culture ¹⁰. With minor adjustments to the protocol, this method should be adaptable to different tumor types or normal tissues.

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Protokół

The following protocol is carried out according to guidelines established by the Department of Laboratory Animal Medicine at the University of North Carolina at Chapel Hill.

1. Prepare the Following Material and Reagents Before Starting

1. Prepare EC media by supplementing 400 ml low glucose (1 g/L D-glucose or LG) Dulbecco's Modified Eagle's medium (DMEM) with 50 ml heat-inactivated fetal bovine serum, 50 ml Nu-Serum IV, 5 ml antibiotic-antimycotic, and the hFGF, VEGF, hEGF, R3-IGF-1, and heparin components from the commercial kit.
2. Prepare 500 ml FACS buffer (0.5% BSA and 2 mM EDTA in PBS); filter through a sterile 0.22 µm filter cup.
3. Sterilize or disinfect dissecting board.
4. Sterilize dissection pins, surgical scissors, and dissectors.

2. EC Isolation (Day 1, ~5 hr)

1. Euthanize mouse with carbon dioxide or other methods compliant with the Institutional Animal Care and Use Committee (IACUC) policies.
   Note: Multiple mammary tumors varying in size (5 - 15 mm in diameter) may develop in a single genetically modified mouse such as C3-TAg, be sure to harvest all of them for TEC isolation. If using tumors that are orthotopically engrafted in mice, pool two to three 1 cm³ tumors for a single EC isolation. Here we use mammary tumors as a demonstration, but the protocol can be modified for other types of tumors.
2. Disinfect mouse by spraying or wiping the mouse ventral side with ample amount of 75% v/v ethanol.
3. Resect tumors with one pair of scissors and dissectors using aseptic techniques in a sterile hood or at a clean bench.
   1. Stretch out and pin the limbs of the mice on a dissecting board. Make a midline ventral incision with the scissors without opening the peritoneum. Dissect laterally between the skin and the peritoneum towards the mammary glands where tumors are located. Do not open the peritoneum.
   2. Excise only tumor tissue from the mammary glands, leaving out normal mammary margins. Carefully trim off non-tumor tissues such as skin and muscles, and place dissected tumors in a conical tube containing 30 ml of LG-DMEM on ice.
4. Bring tumor samples to tissue culture hood; wash tissues with sterile LG-DMEM 1 - 2 times.
5. Transfer tumors from the conical tube to a sterile tissue-culture petri dish, add 2 ml of LG-DMEM in the dish, and mince with a pair of sterile scissors into pieces <5 mm.
6. Add 5 ml of collagenase (stock = 2 mg/ml in Hank’s Balanced Salt Solution, hereafter HBSS), 1 ml of dispase (stock = 2.5 U/ml in HBSS) and 75 μl of deoxyribonuclease (stock = 1 mg/ml in PBS) into the petri dish. Total volume is now ~10 ml.
7. Transfer the collagenase/tissue mix from the petri dish to a tissue-dissociator tube and run on a tissue dissociator for 60 sec twice (pre-set dissociation program on the dissociator: 1,270 total rounds per run). Incubate with light shaking on a shaker for 75 min at 37 °C.
8. Filter digested tissue through a 100 μm cell strainer over a 50 ml conical tube. Rinse filter with 5 ml of FACS buffer to wash any remaining cells. Spin at 280 x g for 5 min and carefully aspirate the supernatant without disturbing the cell pellet.
9. Dilute 1 ml of stock RBC lysis buffer (10x) in 9 ml of sterile water. Lyse red blood cells with 10 ml of lysis buffer (1x), and immediately spin 5 min at 280 x g. Note: This step can be skipped if little blood is visible.
10. Resuspend in 10 ml of FACS buffer. Mix 10 μl of cell suspension with 10 μl of trypan blue, and count live cells using a hemocytometer .
11. Resuspend cells at ~10⁷ cells/100 μl. Add 10 μl of FcR block per 100 μl of cell suspension, and incubate on ice for 10 min.
12. Add rat-anti mouse PE-conjugated CD31 antibody according to Table 1. Incubate on ice for 10 - 15 min and flick tube occasionally.
13. Add 10 ml of FACS buffer to the tube and spin at 280 x g for 5 min. Carefully remove the supernatant, and wash the cell pellet again with 5 ml of FACS buffer. Centrifuge to pellet cells. Aspirate supernatant without disturbing cell pellet.
14. Add FACS buffer and anti-PE microbeads according to Table 2. Incubate on ice for 10 - 15 min. Flick tube occasionally.
15. Add 10 ml of FACS buffer and spin samples at 280 x g for 5 min; wash once with 5 ml of FACS buffer and centrifuge again. Aspirate supernatant without disturbing pellet.
16. Bring volume to 300 μl in FACS buffer. Spin through 35 μm cell-strainer capped tube 5 min at 280 x g. Use 2 tubes and a larger volume if needed.
17. Set up magnetic multistand and magnetic columns in hood, attach a column to the separator and equilibrate the column with 2 ml of FACS buffer.
18. Aspirate the supernatant and resuspend the cell pellet in 0.5 - 1 ml of FACS buffer.
19. Pass the cell suspension through the equilibrated magnetic column.
20. Wash the column three times with 2 ml of FACS buffer, and collect flow-through (FT) in a 15 ml tube (FT fraction).
21. Take the column off the separator and elute with 2 ml of FACS buffer into another 15 ml tube (eluate fraction). Use plunger to ensure all cells are off the column. Repeat the elution two more times with 2 ml of FACS buffer each time.
22. Spin the eluate at 280 x g for 5 min.
23. Remove the supernatant and resuspend the pellet in 10 ml of EC media.
24. Equally divide the eluate fraction (~ 6 ml) into 10 cm gelatin-coated dishes. For three 1 cm³ tumors, plate eluted cells in at least four plates. Alternatively, plate eluted cells at different concentrations in multiple plates (e.g., seed 0.5 ml, 1 ml, 1.5 ml, and 3 ml of the eluate into four plates) to ensure that at least one plate is sparsely seeded with eluted cells. Check that the confluency of the attached cells is at ~1% the next day (i.e., approximately 1.0 - 2.0 X 10⁵ attached cells).
    Note: The cells need to be plated sparse so that EC colonies can form without being contaminated by other cell types.
25. Plate FT fraction in one 10 cm dish to let cells recover O/N. Check that the plate is 80 - 100% confluent the next day. Freeze down the cells (-80 °C) in 250 µl cell-freezing media in a cryotube and store them in liquid nitrogen the next day. Note: FT fraction can be used for tumor cell isolation at a later stage and/or as a negative control for EC gene expression analysis of the isolated EC clones.
26. Change media every 2 - 3 days. Colonies start to form after 7 - 10 days. Small EC colonies can be identified as early as day 3. Mark the colonies with a fine-tip marker on the bottom of the dish.
27. Scrape off non-specific cells surrounding the identified colonies with a sterile 200 µl pippet tip.

3. Colony Selection Using Cloning Rings

1. Change media every 2 - 3 days. EC purity can be checked by LDL (DiI-Ac-LDL) addition at about 5 - 7 days. Add 50 µl of LDL per 10 ml EC medium and incubate for 3 - 4 hr before checking the cells under fluorescence microscope.
   Note: Multiple LDL⁺ EC clones could be observed at ~day 7.
2. Start harvesting EC colonies when they reach diameters of 3 - 5 mm in size. (Choose big colonies that are packed with small LDL⁺ cells for best results.)
3. Before harvesting EC with cloning rings, pre-coat a few 6-well plates with 0.5% gelatin. Aspirate gelatin, add 2 ml of EC media to each well, and keep the plates in an incubator until needed.
4. Scrape off non-EC on the edges of the colonies to make sure no other cell types will be trapped within the cloning ring.
5. Using a phase-contrast microscope (4X or 10X objective), outline with a fine-tip marker on the bottom of the culture dish the areas containing EC colonies.
6. Wash the plate with 10 ml of PBS and leave a very thin layer of PBS in the plate when aspirating. (Important: a small amount (~0.5 ml) of PBS will keep cells alive during the cloning-ring procedure; also, tissue adhesive needs water to bond.)
7. Choose a cloning ring of appropriate size. Use a pair of dissecting forceps to pick up a ring, and with a 10 µl pipet tip evenly apply a small amount of tissue adhesive onto the cloning ring.
   Note: use only minimal amount (~0.2 μl for a small ring) of tissue adhesive on cloning rings, and make sure tissue adhesive is spread out evenly around the bottom surface to ensure good sealing. Excessive tissue adhesive produces heat and forms films that may kill cells.
8. Place the cloning ring over the EC colony. Gently press down the cloning ring to glue the ring onto the plate. Ensure that the colonies are not dried out before gluing the ring.
9. Immediately pipet 25 µl of enzymatic cell detachment solution into the cloning ring and incubate ~1 min or until the cells are loosely attached.
10. Pipet cells in the cloning ring drop-wise into one well of a 6-well plate containing pre-warmed EC media. (Important: Do not shake the plate to disperse cells; EC prefer to grow in tight clusters.) Wash the cloning ring with 50 - 100 µl EC media to collect as many cells as possible, and transfer all washes into the same 6-well.
11. If some colonies in the 10 cm dish are too small to harvest, add 10 ml fresh media, let the colonies grow for a few more days, and repeat the cloning ring procedure again.
12. Grow harvested colonies in 6-well plates until 80 - 100% confluent, and transfer cells to 3 wells of a 6-well plate, before expanding them in a 10 cm tissue culture dish. Scrape off contaminant cells. Repeat another round of cloning ring procedure (Steps 3.5 - 3.10) if necessary. Keep cells relatively confluent (~60 - 70%) when expanding. EC may stop growing if plated too sparse.
13. Characterize EC by FACS, staining, PCR, etc. Note that Dil-Ac-LDL is fluorescent and may interfere with PE or other fluorescent antibodies for FACS.

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Wyniki

EC represent only minor a fraction of the total cell population in most adult tissues ¹¹. It is therefore important to fully digest the harvested tissue into a single-cell suspension that ensures the maximal release of EC from extracellular matrix (ECM) and connective tissues. In our experience, CD31-mediated immunomagnetic selection only provides enriched but not pure EC fractions; therefore, another crucial step is the physical removal of co-purified non-EC and selection/expansion of EC colonies using clonin...

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Dyskusje

Due to the difficulties in obtaining pure primary TEC cultures, many in vitro studies substitute TEC with commercially available EC lines or primary EC such as human umbilical vein EC (HUVEC) ¹³. However, these EC populations from normal tissues may only serve as a proxy for TEC which differ markedly from their normal counterparts. For example, TEC are phenotypically and functionally abnormal in vivo and some of these abnormalities may be transmittable in vitro ^14-18. TEC ...

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Materiały

Name	Company	Catalog Number	Comments
Antibiotic-Antimycotic	Sigma-Aldrich	A5955
Dulbecco's Modified Eagle's medium (1 g/L D-glucose) (LG-DMEM)	Gibco	11885-084
EGM-2 Bullet Kit	Lonza	CC4176	Not all components used
Fetal bovine serum (Hyclone)	Thermo Scientific	SH30071.03	Heat inactivated at 56 °C for 30 min
Nu-Serum IV	Corning	CB-51004
Hank's Balanced Salt Solution (HBBS)	Gibco	14175-095
Phosphate-buffered saline (PBS)	Gibco	14190-144
FACS buffer			0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 μm filter
75% v/v ethanol for disinfection
Anti-PE microbeads	Miltenyi Biotech	130-048-801
Bovine serum albumin (BSA) fraction V, 7.5%	Gibco	15260-37
Cell freezing media (Bambanker)	Wako Chemicals	302-14681
Collagenase type II	Worthington Biochemical	LS004176	Make stock concentration 2 mg/ml in HBSS
Deoxyribonuclease I (DNase)	Worthington Biochemical	LS002004	Make stock concentration 1 mg/ml in PBS
Dil-Ac-LDL	Biomedical Technologies	BT-902
EDTA, 0.5M, pH 8.0	Cellgro	46-034-CL
Enzymatic cell detachment solution (Accutase)	Sigma-Aldrich	A6964-100ML
Gelatin, 2% in water, tissue culture grade	Sigma-Aldrich	G1393-100ML	Dilute in PBS to make 0.5% gelatin solution
Mouse FcR Blocking Reagent	Miltenyi Biotech	130-092-575
Neutral protease (Dispase)	Worthington Biochemical	LS02104	Make stock concentration 2.5 U/ml in HBSS
PE-rat anti-mouse CD31 antibody	BD Pharmingen	553373
RBC lysis buffer (BD Pharm Lyse)	BD Pharmingen	555899
Sterile water
Trypan blue, 0.4 %	Life Technologies	15250-061
10 mm tissue culture dishes	Corning
15 ml conical tubes (sterile)	Corning
50 ml conical tubes (sterile)	Corning
6-well tissue culture plates	Corning
Tissue-dissociator tubes (gentleMACS) C tubes)	Miltenyi Biotech	130-093-237
Cell Separator  (MidiMACS)	Miltenyi Biotech	130-042-302
Cell strainer 100 μm	Corning	352360
Cloning rings (assorted sizes)	Bel-Art Products	378470000
Cryotubes	Thermo Scientific
Dissecting board			Sterilize or disinfect with 75% v/v ethanol before use
Dissecting forceps and scissors			Sterilize before use
Dissecting pins 2"			Sterilize before use
FACS tubes with 35 μm filter cap	Corning	352235
Filter cup (Stericup, 0.22 μm)	Millipore	SCGPU05RE
Fine-tip marker
Hemocytometer
LS Columns	Miltenyi Biotech	130-042-401
Magnetic Multistand	Miltenyi Biotech	130-042-303
Tissue adhesive (Vetbond)	3M	1469SB
Centrifuge	Eppendorf	5810R	Or a centrifuge with similar capacity for 15 ml and 50 ml conical tube centrifugation
Tissue culture hood
Tissue dissociator (gentleMACS)	Miltenyi Biotech	130-093-235	Preset program "m_impTumor_01" used for tissue dissociation
Liquid nitrogen freezer
Microplate or rotary shaker
Phase contrast light microscope