Application of cryobiopsy to morphological and immunohistochemical analyses of xenografted human lung cancer tissues and functional blood vessels

Abstract

Assessment of tissue specimens obtained with common immersion-fixation followed by dehydration (IMDH) is affected by artifacts, which hinder precise evaluation of the histology and microenvironment of tumor tissues. The technical characteristics of cryobiopsy and in vivo cryotechnique (IVCT) where target organs are directly cryofixed in vivo are still unknown in practical examinations of tumor histopathology and microenvironment. Three lines of human lung cancer cells were subcutaneously injected to the dorsal flank of nude mice, and paraffin sections and cryosections of produced tumors were prepared with cryobiopsy, IVCT, the quick-freezing of the fresh resected tumor tissues, or IMDH. Histological comparison among different methods was conducted, and immunolocalization of immunoglobulin M (IgM), intravenously injected bovine serum albumin (BSA), and vascular endothelial growth factor (VEGF) were examined. With both the cryobiopsy and IVCT, cellular morphology and open blood vessels with flowing erythrocytes could be observed without artificial shrinkage, and the volume of blood vessels was not affected by a vascular collapse, which was observed after tissue-resection. In addition, with cryobiopsy and IVCT, IgM was well preserved in functional vessels with blood flow, which could be observed with injected BSA, and the volume of IgM-immunopositive blood vessels was significantly associated with the expression of VEGF. Cryobiopsy could be useful for histological examination of human tumors without morphological artifacts associated with IMDH. Furthermore, it allows direct examination of functional blood vessels and related signaling molecules, thereby providing a better evaluation of the human tumor microenvironment for clinical application.