In this report, we investigated the in vivo cell biology of cancer cells during immune rejection. The use of nestin-driven green fluorescent protein (ND-GFP) transgenic mice as hosts, in which nascent blood vessels express GFP, and implanted dual-color mouse mammary tumor 060562 (MMT) cells, in which the cytoplasm expresses red fluorescent protein (RFP) and the nuclei express GFP, allowed very important novel observations of angiogenesis and subcellular death pathways during immune rejection of a tumor. Nascent blood vessels did not form in the initially-growing mouse mammary tumor in ND-GFP immunocompetent mice. In contrast, in ND-GFP immunodeficient nude mice, numerous GFP-expressing nascent blood vessels grew into the tumor. The results suggest that insufficient nascent tumor angiogenesis was important in tumor rejection. During immune rejection, the cancer cells deformed their cytoplasm and nuclei, which were readily imaged by RFP and GFP, respectively. The nuclear membrane of the cancer cells ruptured, and chromatin extruded during partition of cytoplasm and nuclei. T lymphocytes infiltrated into the initially-growing tumor in the nestin-GFP transgenic immunocompetent mice. The cytotoxic role of the sensitized T lymphocytes was confirmed in vitro when they were co-cultured with MMT cells. The CD8a-positive lymphocytes attached to the cancer cells and caused nuclear condensation, deformation, and partition from their cytoplasm, similar to what occurred in vivo. The color-coded subcellular fluorescence-imaging model of immune rejection of cancer cells can provide a comprehensive system for further testing of immune-based treatment for cancer.
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