Abstract
e23074 Background: The purpose of the study was to assess stress-induced metabolic changes in СD4+ T-lymphocytes among tumor-infiltrating lymphocytes (TILs) according to ultrastructural morphometric parameters. Methods: Samples (n = 12) of colon adenocarcinoma, corresponding resection line tissues and blood of patients and healthy donors were studied. After grinding the samples and PBMC isolation using the density gradient, CD4+ T-lymphocytes separation with magnetic columns was performed using CD4 MicroBeads, human (MACS Miltenyi Biotech). The pellet obtained after centrifugation was embedded in agarose and prepared for transmission electron microscopy. Mitochondria images on ultrathin sections of Т-lymphocytes were randomly selected for the analysis. The total number of mitochondria studied were 71 (tumor), 78 (normal tissue), 78 (blood of patients), 80 (blood of healthy donors). For each image the unchanged cristae area was determined as a percentage of the total area of mitochondria cross section. Results: We found no differences in the structures of mitochondria of blood lymphocytes in patients and in healthy donors. Comparison of parameters of CD4+ Т-lymphocyte mitochondria in the colon unchanged tissues and in the blood of patients did not show significant differences indicating minimal damage during TILs` isolation from the tissues. Electron microscopy of TILs derived from tumor tissue showed more frequent apoptotic and necrotic death of lymphocytes, as well as some mitochondrial swelling with changes in the shape and the number of cristae compared to the norm. Statistical analysis revealed significant decrease in the relative area of unchanged cristae: 57% in tumor against 73% in the normal tissue (p < 0.0001, χ2Test). Conclusions: The results suggest that stress-induced changes are developed in tumor-infiltrating CD4+ T-lymphocytes, and these changes apparently enhance their death as demonstrated by ultrastructural characteristics of mitochondria.
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