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

Summary

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.

Abstract

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.

Introduction

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

Protocol

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.
....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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