The In ovo CAM-assay as a Xenograft Model for Sarcoma

Podsumowanie

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Streszczenie

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.

This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

Wprowadzenie

Sarcoma is a rare tumor of the connective tissues with a high mortality due to therapy resistance^1,2. Progress in patient survival is hampered by their low annual incidence, their broad diversity, and the fact that sarcoma cells are reported to be hard to culture in vitro^3,4.

The use of cultured cells for preclinical therapy evaluation has revealed that new, apparently active molecules in vitro do not always reflect results in the clinical setting. Furthermore, genome aberrations revealed by gene expression arrays are not always correlated to tumor behavior characteristics in the individual patient^5-7. In order to try and solve these problems, personalized medicine has gained in importance, which is reflected in the increased search for xenograft models^8-12.

An in vivo assay has the advantage of reflecting the complex interplay between cancer cells and the host tissue environment in solid tumors, necessary for cancer proliferation and invasion¹³. Currently we study the use of the Chorio-Allantoic Membrane assay (CAM-assay) as a reproducible xenograft model for sarcoma^14,15. This assay is widely used for the study of tumor angiogenesis^16,17. In literature, however, we have found different protocols for this assay, while other studies observed a marked difference in growth or angiogenesis according to different protocols^18,19.

In this article we investigate the effect of varying conditions of the CAM-assay on cell behavior using tumor grafts, tumor-derived single cell suspensions and established sarcoma cell cultures.

Protokół

See Figure 1 for an overview.

Tumor material

1. Obtaining and Preparing Tumor Samples

For the use of patient material, approval of the Ethical Committee is necessary, and informed consent has to be obtained from the patient.

1. Harvest representative material (minimum 1 cm³) at the time of intervention, either a biopsy or a resection of a sarcoma. The proper site of biopsy is defined using dynamic-contrast MRI.
2. Place the material immediately in a sterile vial containing DMEM supplemented with 1,000 U Penicillin and 1 mg/ml streptomycin.
3. Transport the vial immediately (30 - 90 min) to the lab.

All following procedures are performed under laminar flow.

4. Pour the contents of the vial into a sterile Petri dish.
5. Put the tumor sample into small parts of maximally 1 mm³ using a sterile scalpel. Remove all calcified parts as they tend to impair further processing of the tissue.
6. Randomly take 10 tumor grafts for application on the CAM.

2. Preparation of Tumor-derived Single Cell Suspensions

Approximate duration: 3 hr.

1. Weigh the remaining tissue of the sample.
2. Put 2 - 4 g of tissue in a dissociator tube, more than one tube may be required to digest all the remaining tumor tissue.
3. For digestion of 2 - 4 g of tissue, add 2.5 ml collagenase 2 solution (500 U/ml RPMI 1640) and 2.5 ml DNase solution (22 KU/ml RPMI 1640).
4. Process the tissue according to the dissociator h_tumor protocol. This involves 2 cycles of incubation at 37 °C in CO₂ incubator while the tube is shaken gently for 30 min.
5. Filter the remaining suspension through a 150 μm cell strainer.
6. Centrifuge the lysate at 1,000 rpm for 5 min.
7. Remove the supernatant.
8. Resuspend the pellet in 10 ml of erythrocyte lysis buffer (ELB), allowing 10 min of incubation with regular suspension.

Note: erythrocyte lysis buffer is manufactured as follows: Add 0.037 g EDTA, 0.99 g K₂HPO₄, 8.29 g NH₄Cl to 1,000 ml aqua, use 10 N NaOH to adjust the pH to 7.3, filter under pressure through a 0.22 μm filter. Store at 4 °C.

9. Add 25 ml of culture medium (DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin) to stop the reaction.
10. Centrifuge the suspension at 1,000 rpm for 5 min. The color of the pellet should be white now.
11. Remove the supernatant.
12. Add 5 ml of medium to the pellet. Filter the suspension through a 70 μm cell strainer.
13. Count the number of live cells/ml by means of an automatic cell counter.

3. Cancer Cell Lines

SAOS2 osteosarcoma cells (ATCC number: HTB-85) expressing enhanced green fluorescent protein were electroporated by peGFP-C1 vector using the Cell Line Nucleofector Kit V according to the manufacturer's protocol. To establish stable cell lines, transfected cells were selected in G418 (1 mg/ml) for 4 weeks in line with previous established protocol²⁰. In addition sorting was performed by fluorescence activated cell sorting (FACS) according to the manufacturer's instructions.

Cells from the SW1353 chondrosarcoma cell line (ATCC number: HTB-94) or freshly prepared tumor single cell suspensions are labeled using a red fluorescent lipophilic membrane stain.

1. Remove the cells from the culture flask in a classical way using Trypsin (0.5% wt/vol) ethylenediaminetetraacetic acid (EDTA; 0.2% wt/vol) solution.
2. Centrifuge the cell suspension for 5 min at 1,000 rpm.
3. Remove the supernatant.
4. Add 1 ml of serum-free medium and 5 μl DiI (Vybrant Red)/10⁶ cells.
5. Wrap the vial in aluminum foil and put it in the CO₂ incubator for 10 min at 37 °C.
6. Centrifuge again for 5 min at 1,000 rpm and remove the supernatant.

Chorioallantoic membrane assay

1. Egg Incubation

1. Fertilized eggs from a local commercial hatchery guaranteeing 95% fertilisation of the eggs are required.
2. Fertilized eggs can be stored at RT up to 10 days.

Note: before starting incubation, dirt, feathers and excrement are carefully removed from the eggshells mechanically by dry wiping with paper towels, which have a rough rather than a soft surface structure. Wiping the eggs with 70% denatured ethanol or any other cleaning reagent significantly reduces the survival rate of the chick embryos.

3. Put the eggs in the incubator on one side, marking the upper surface. The day on which the eggs are put in the incubator is designated day 0 of embryonic development.
4. Set the incubation temperature at 37.8 °C, and the humidity at 47%. Make sure the automatic rotation option is switched off.

2. Opening of the Eggs

Duration: ± 60 min for 25 eggs.

On development day 3, the eggs are opened under laminar airflow. Opening the eggs at a further developmental stage results in damage to the CAM, as the membrane tends to stick to the shell. An infrared lamp is used to keep the eggs warm during the procedure. To improve sterility, we recommend the use of hand gloves.

1. Place the egg in the eggcup, with the marked side facing upwards.
2. Lower the level of the CAM by removing two ml of albumen through an 18 G needle inserted at the tip of the egg. If the needle is blunt after perforating several eggshells, replace the needle. If not, too much force has to be exerted, resulting in damage to the egg yolk or fracture of the shell. Sometimes the needle is blocked by the chalazae, one of 2 spiral bands suspending the yolk in albumen. This can be solved by gently pushing the plunger back down until the block is resolved. If egg yolk is sucked into the syringe, replace the needle as well as the syringe. Sometimes the embryo dies after this event. If an insufficient amount of albumen is removed, the CAM will be damaged when the shell is removed.
3. Apply a semipermeable adhesive film at the marked upper side of the shell in order to prevent spilling of shell particles onto the CAM while cutting the window.
4. Make an introduction hole with a small awl at the surface of the egg.
5. Cut a window of 1 cm² in the shell, using a pair of sterile sharp-pointed surgical scissors.
6. The embryo's pulsating heart and adjoining vessels can be observed at the surface of the egg yolk. Remove the non-fertilized eggs or dead embryos. An interrupted aspect of the blood vessels characterizes dead embryos.
7. Seal the window with a semipermeable adhesive film. It is not necessary to cover the puncture site, unless the shell has cracked during insertion of the needle.

3. Inoculation Procedure

Duration: ± 1 hr per cell line.

On chick embryonic development day 9, the eggs are inoculated under laminar airflow.

Evaluation of cancer cell spreading across the CAM requires containment of the cells to a limited surface during inoculation. Matrigel is an extracellular matrix (ECM) gel from the Engelbreth-Holm-Swarm mouse sarcoma frequently used in experimental cell culture conditions. It is a fluid when cooled, but will undergo thermally activated polymerization when brought to 20 - 40 °C, thus forming a stable gel. During the experiments, Matrigel is kept in a box containing melting ice.

Note: We insist not to use perforated coverslips. We observed attachment and growth of grafted cancer cells onto the plastic disc thus biasing growth potential of the cells on the membrane itself.

1. Resuspend a cell pellet in cooled ECM gel at a concentration of 10⁶ cells/100 μl ECM gel. Keep this solution on melting ice.
2. Excise part of the semi-permeable membrane with a pair of sterile surgical under laminar airflow. If the adhesive film is removed completely, this will result in a larger proportion of embryonic death due to contamination.
3. Touch the CAM right below the shell window lightly with a sterile glass rod, in order to remove the epithelial cell layer. Touching the CAM with a sterile glass rod proves to be less traumatic than using a scalpel n° 11, which requires more handiness and experience. Small hemorrhages may occur.
4. Adding tumor material.
5. For tumor grafts: gently lower one piece of tissue onto the CAM with a pair of sterile forceps, without squeezing it.
6. For the ECM gel procedure: Add 4x 25 μl of the cell-ECM gel suspension to the CAM, on top of each other, allowing the ECM gel to polymerize in between the applications. In this way an adherent plaque containing the cancer cells is created.
7. Seal the shell window again with a semipermeable adhesive film.

4. Imaging and Harvesting of the CAM

Duration: 2 hr for 25 eggs.

On chick embryonic development day 16, the CAMs are harvested. Working under laminar airflow is not necessary.

1. Enlarge the window with a pair of surgical scissors. Make sure not to cut too low, otherwise the CAM will be damaged, resulting in bleeding of the injured vessels.
2. Add 300 - 500 μl of PBS^D with a pipette onto the CAM section containing the inoculated material. If no fluorescent probes are used, buffered formaldehyde may be used as this makes the membrane stiffer, facilitating the cutting of the membrane.
3. Make a photograph of the CAM in the egg through a stereo fluorescence microscope, equipped with a digital color camera, using both the GFP or the DSR filter, and no filter. For the tumor grafts, all photographs are made without filters. Illumination from above by LED lights is strongly recommended during photographing (Figure 2).
4. Record tumor cell migration for the labeled cells. The borders of the tumors may be irregular and frayed. Scattered cells may be present near the tumor borders. In some specimens, rows of tumor cells may be observed (Figure 3).
5. Cut the membrane with a pair of sterile scissors at the border lying against the shell.
6. Place the harvested CAM in a sterile Petri dish filled with PBS^D or buffered formaldehyde.
7. Make a photograph of both the upper side and the lower side of the CAM, both with and without filter.
8. Put the membrane in a labeled container with buffered formaldehyde for at least 72 hr.
9. Embed the CAMs in paraffin and prepare them for histological examination. Take care to cut the tumor grafts at the center of the nodule, making sure the cut surface is parallel to the bottom of the cassette. If not, the interaction between the CAM and the tumor graft will not be visible. The same strategy applies to the ECM gel plaques: the surface facing the cutting blade should be perpendicular to the plaque.

5. Histological Evaluation

Hematoxylin-Eosin staining and Ki-67 immunohistochemistry were performed for further clarification, if needed. Immunohistochemistry was performed on formalin-fixed paraffin-embedded tissue sections of 3.5 μm in thickness, using an automated slide stainer according to manufacturer's instructions. A mouse primary monoclonal antibody to Ki-67 (clone MIB-1, dilution 1/100) was used. Heat-induced epitope retrieval was performed using Cell Conditioning 1, and visualization was achieved with the Universal DAB Detection Kit, according to manufacturer's instructions. Dehydratation of the tissue sections was carried out using an automated coverslipper.

For the SAOS2 and the SW1353 cell lines, the number of Ki67-positive and Ki67-negative cell nuclei/100 μm² were counted in three zones: one close to the border of the CAM, one close the surface and one in the center of the ECM gel plaque. This procedure was repeated 6x for each Ki-67 stained slide. The index of proliferation was determined for each slide by dividing the number of Ki67-positive cells by the total number of cells counted.

Necrosis is defined by a loss of fine dispersion of chromatin in the cell nucleus, accompanied by fading of distinct cell margins. For xenografts, necrosis is scored as complete, partial (often at the surface), or absent. Infiltration of tumor cells into the CAM is defined by the observation of tumor cells within the mesoderm of the CAM, and is scored as being present or absent.

Three items are scored for host behavior. Fibroblast infiltration is defined as elongated cells leaving the CAM and invading the tumor graft/plaque, and is scored as being present or absent. Vascular ingrowth can be observed as ingrowth of proliferating chick vessels, characterized by the presence of nucleated chick erythrocytes.

Wyniki

Evaluation of the CAM

Tumor grafts become adherent to the CAM (Figure 2A). Single cell suspensions from patient material frequently display a dried, slightly raised plaque (Figure 2D). After excision of the CAM, marked wrinkling of the membrane occurs (Figures 2E and 2F).

For the commercial cell lines the plaque becomes more opaque in time, indicating cell proliferation. Different cell lines in ECM gel...

Dyskusje

Time of inoculation and harvest

Timing the day of inoculation was performed using SAOS2 in ECM gel (36 CAMs) and varied between embryonic development day 5 and 10.

Before incubation day 9, the CAM was not consistently large enough to support the ECM gel we applied. At harvest, tumor cells sometimes had to be retrieved from the deeper CAM, and some ECM gel samples were lying loose in the albumen or on the CAM. On days 5 and 6, it was hard to avoid putting the tumor ce...

Materiały

Name	Company	Catalog Number	Comments
Cell Line Nucleofector Kit V	Amaxa	VCA-1003
collagenase 2 solution (500 U/ml RPMI 1640)	Sigma Aldrich	C6885
DMEM	Invitrogen	41965-039
DMSO	Sigma	D8418
Dnase solution	Sigma Aldrich	DN25
G418	Invitrogen	11811031
Matrigel	Sigma-Aldrich	E1270
mouse primary monoclonal antibody Ki67	Dako Denmark	MIB-1
Paraformaldehyde	Fluka	D76240
PBS	Invitrogen	20012019
PBSD	Invitrogen	14040083
peGFP-C1 vector	Clontech	632470
Penicillin/streptomycin	Invitrogen	15140163
RPMI	Invitrogen	22409-015
Trypsin-EDTA solution	Invitrogen	25300054
Vybrant cell-labeling DiI	Lifetechnologies	22885
Countess Automated Cell Counter	Invitrogen	C10227
digital color camera	Leica	DFC 340 FX
Digital Egg Incubator	Auto Elex Co	R-COM 50
FACS	BD Biosciences	FACSAriaIII
Gentlemacs C-Tube	Miltenyi Biotech	130-093-237
Gentlemacs Dissociator	Miltenyi Biotech	130-093-235
Gentlemacs Dissociator User Manual containing h_tumor protocol	Miltenyi Biotech
[header]
semipermeable adhesive film (Suprasorb F)	Lohmann&Rauscher	20468
stereo fluorescence microscope	Leica	M205 FA
Tissue-Tek Film automated Coverslipper	Sakura	6400
ultraView Universal DAB Detection Kit	Ventana Medical Systems Inc	760-500
Ventana Automated Slide Stainer	Ventana Medical Systems	Benchmark XT