Tumors were obtained after approval from the institutional ethics review board of All India Institute of Medical Sciences (AIIMS), New Delhi, India (IEC-299/01.06-2018) and the study followed the tenets of The Declaration of Helsinki. All patients provided written informed consent.
Approval to work on fertilized chicken eggs in the study was obtained from the ethical and safety committee of Shivaji College, University of Delhi, New Delhi, India (Bioethics and biosafety committee report, SCS2019).
Egg Procurement and Preparation
An overview of the protocol has been described in Figure 1.
Fertilized eggs of Gallus gallus domesticus were procured from Chattarpur Farms, New Delhi, India. In the research laboratory, these eggs were cleaned using sterile water and paper towels. Following this, the eggs were maintained in an upright position under suitable conditions of 37.5°C temperature and 55-60% relative humidity in order to induce embryogenesis. The study was carried out after approval from the institutional ethical and safety committee of Shivaji College, University of Delhi, New Delhi, India (Bioethics and biosafety committee report, SCS 2019).
The CAM assay was performed between the chick embryonic development day (EDD) 9 and 17. On EDD 9, the CAM tissue was identified. Further, the eggs were cleaned with distilled water, followed by which the air sac was punctured using a 19 G needle, and a suction force was applied for the dropping of CAM. A window of 1.5 cm diameter was established on the upper surface of the eggs under aseptic conditions, and all the embryos were examined for any signs of local bleeding. Next, sterile silicon -o- ring (inner and outer diameter 8mm and 10 mm respectively) was placed onto the CAM in an area with visible vascularization.
Tumor Procurement and Preparation
Fresh tumor tissues of Choroidal Melanoma (CM) and Retinoblastoma (RB) were obtained from two patients undergoing enucleation from the Operation Theatre of Dr. R.P. Centre, All India Institute of Medical Sciences (AIIMS), New Delhi after written and informed consent from donor patients and approval from the institutional ethics review board of AIIMS, New Delhi, India (IEC-299/01.06-2018). The samples were transported from the Department of Pathology in isotonic saline solution at ambient temperatures of 18-20°C.
The tumor sample procured from the patient with Choroidal melanoma was divided into three tissue segments (replicates) of approximately 4-5 mm length, and the same step was followed for the tumor sample procured from the patient with Retinoblastoma. The replicates were excised with minimal disruption of the ocular architecture using a scalpel and blade. The rest of the tumor tissue was formalin-fixed and paraffin-embedded for light microscopy and immunohistochemistry. The patient-derived xenografts were then subjected to CAM assay in separate set ups. On EDD 10, each excised replicate of CM and RB was placed separately in the middle of a sterile silicon -o- ring on the CAM of the egg after 45-60 minutes of enucleation. The egg windows were resealed with sterile tape.
Patient-Derived Xenografts and CAM Assay
The six inoculated eggs containing the patient-derived xenograft were placed back in the incubator, maintaining them in an undisturbed state for 7 days until EDD 17. On EDD 17, the chick was euthanized by inducing hypothermia. Following this, the CAM tissue bearing the patient-derived xenograft was extracted and washed with PBS to remove the remnants of yolk and extraembryonic membranes. The observations were then made under a stereoscopic microscope and photographs were taken. Shortly after this, the extracted tissue bearing the tumor sample was fixed in 3.7% paraformaldehyde for further processing.
Following fixation and processing of the CAM tissue bearing the tumor, standard protocols were utilized to accomplish Hematoxylin and Eosin (H/E) staining on the sections. Sections were mounted and imaging was performed using light microscopy. H/E stained sections were then evaluated by an ophthalmic pathologist (S.S) and two observers. The tumor invasion was shown to be represented by the occurrence of invasive cancer cells in the mesoderm of CAM on histological sections analysis.
Paraffin-embedded and formalin-fixed tissue segments were mounted on poly L lysine microscope adhesion slides. Next, deparaffinization was performed by utilizing xylene, followed by which, rehydration was achieved using a series of graded alcohols. After this, antigen retrieval was performed by incubating tumor sections for approximately 20 minutes at a temperature of 90°C in a citrate buffer of pH 6.0. The slides were then allowed to cool, after which they were washed with Tris-Buffered Saline of pH 7.5 and the endogenous peroxidase activity of the tumor sections was stopped by incubating them in H2O2 (0.3% v/v) for 20 minutes. The retinoblastoma xenograft tissue and associated CAM tissue sections were then incubated with monoclonal anti- synaptophysin antibody (MA5-14532, Thermo Fisher) and anti-Ki-67 antibody (MA5-14520, Thermo Fisher) at dilution of 1:200. The immunoreactive signals were identified using the substrate DAB peroxidase. Hematoxylin was used as a counterstain for 30 seconds, following which the tissue sections were dehydrated and DPX was utilized for mounting them. The results were demonstrated using light microscopy.