A macroscopic postmortem analysis included examination of the external surfaces, appearance of primary tumor nodes, thoracic condition, and abdominal and pelvic cavities with their associated organs and tissues. For further histological evaluation, specimens of tumor nodes from each animal were collected during autopsy and fixed in 10% neutral-buffered formalin, dehydrated in ascending ethanols and xylols, and embedded in HISTOMIX paraffin (BioVitrum). Paraffin sections (5 μm) were stained with hematoxylin and eosin, microscopically examined, and scanned. Tumor sections for immunohistochemical studies (3 to 4 μm) were sliced on a Microm HM 355S microtome (Thermo Fisher Scientific) and further deparaffinated and rehydrated; antigen retrieval was carried out after exposure in a microwave oven at 700 W. The samples were incubated with the CD8-specific antibodies (M3164, Spring Bioscience) according to the manufacturer’s protocol. Next, the sections were incubated with secondary HRP-conjugated antibodies (Spring Bioscience detection system), exposed to DAB (3, 3-diaminobenzidine) substrate, and stained with Mayer’s hematoxylin. Images were obtained using an Axiostar Plus microscope equipped with an Axiocam MRc5 digital camera (Zeiss, Germany) at 10×, 20×, and 40× magnifications. Gross examination of tumors included evaluation of size of the tumor node, presence of a capsule, and presence of necrosis and hemorrhages. Microscopic examination of tumors included evaluation of histopathological changes in tumor tissue in terms of necrosis and apoptosis, presence of mitoses, and presence of CD8 lymphocyte infiltration.

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