Investigations in the present study were performed in accordance with the principles outlined in the Declaration of Helsinki and approved by Kaunas Regional Bioethics Committee (P1-BE-2-34/2007), Kaunas, Lithuania. LSCC tissue samples were acquired in accordance with the protocol approved by the Institutional Review Board of Lithuanian University of Health Sciences (LUHS), Kaunas, Lithuania. Written Informed Consent was obtained from the patients before surgery and patients’ identifiers were removed to ensure anonymity.
Twelve male patients with histologically verified LSCC participated in this study. The mean age of the patients was 54±7 years. Distant metastases of LSCC were diagnosed in 5 cases. Four patients passed away during the 5 years follow up period after LSCC diagnosis. Data about the differentiation grade and the stage of the LSCC is given in Table 4.
The five-year follow up data from the group of LSCC patients starting from the experimentation day were analyzed according to the differentiation grade and the stage of the disease. All patients were evaluated for the recurrence of LSCC and distant metastasis every 3 months during the first 2 years and every 6 months during the next 3 years. Those patients who did not survive to the end of experimentation were noted too.
Incubation and egg opening
Fertilized hen eggs (Cobb-500) (N=20) were incubated at 37.7 °C temperature and 59-60 % relative humidity for each experiment, in total 240 ones. On the day 3 of incubation albumen was removed and window in the egg shell was opened. Embryos that showed signs of vitality were prepared for further incubation by covering egg-shell windows with the sterile transparent tape in the same conditions for 96 to 144 hours when the fresh LSCC tumor tissues were implanted.
LSCC tissue implantation onto the CAM, tissue sampling and histological evaluation
Fresh 219 LSCC tissue samples were obtained from 12 male patients who underwent laryngeal surgery because of LSCC in the Department of Otorhinolaryngology, LUHS. Tumor samples were placed in isotonic saline solution for transportation to Department of Histology and Embryology, LUHS, and then implanted onto CAMs within 45 minutes. Each patient’s LSCC tissue sample was sliced in small pieces of about 0.8 mm³ and gently placed on the outer surface of the CAM (1 piece per egg) 13.
In vivo biomicroscopy was performed for each embryo every 48, 72, 96, 120, 144 and 168 hours after tumor implantation. The random two eggs at each of the reported hours were injected with 10 µl of a 20 mg/ml 70- kDa fluoresceinated anionic dextran (Eugene, OR, USA) in phosphate-buffered saline (PBS) into the biggest apparent vessel of the CAM14. The newly formed micro-vascular network on the CAM was evaluated with OLYMPUS SZX 16 stereomicroscope (Olympus Life Science Europa GmbH, Hamburg, Germany). CAMs with the ingrowing implants were excised, fixed in formalin, cut out and embedded into paraffin blocs for the period of 5 days. The embedded membranes were sliced and stained with hematoxylin and eosin (H&E) for histological examination. Histological evaluation of the samples stained with H&H was performed with the cold light microscope OLYMPUS BX40F4 (Olympus Opticae co. LTD., Japan) under 10x magnification using CellSensDimention1.9 Digital Imaging Software for Research Applications (Olympus Corporation of the Americas, USA). The thicknesses of the CAM and of the chorionic epithelium per constant length of the CAM section were measured under the implanted tumors on the images obtained with Olympus digital camera (Olympus U-CMAD3, Philippines).
Twenty-two CAMs that proceeded under the same protocol, however without LSCC implantation constituted the control group.
Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 20.0. (Armonk, NY: IBM Corp. Software). Data were presented as the mean ± standard deviation (SD). The Student’s t test was used for testing hypothesis about equality of the mean. The size of the differences among the mean values of the groups was evaluated by estimation of type I and type II errors (α and β) of the tests. The difference was considered to be significant if β≤0.2 and α=0.05. The level of statistical significance by testing statistical hypothesis was 0.05.