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Purpose Heat shock protein 70 (Hsp70) was detected on the cell membrane of human tumor cell lines, but not on normal cells. Here we studied Hsp70 membrane expression as a target for natural killer (NK) cells on tumor material and control tissues of head-and-neck cancer patients. Methods and materials Membrane-bound Hsp70 was determined by flow cytometry on single-cell suspensions of tumors and the corresponding normal tissues of head-and-neck cancer patients. The cytolytic activity of NK cells against Hsp70-positive tumor cells was measured in a standard cytotoxicity assay. Results In total, 54 of 74 primary tumors were found to be Hsp70 membrane-positive (73%); tongue/mouth, 21 of 24 (88%); oropharynx, 13 of 20 (65%); hypopharynx, 3 of 6 (50%); larynx, 8 of 11 (73%); trachea 1 of 2 (50%); esophagus, 4 of 5 (80%); lymph node metastases, 4 of 6 (67%). The corresponding control tissue was negative for membrane-bound Hsp70. Biopsies (6 of 6) of patients after in vivo γ-irradiation (fractionated 5 × 2 Gy) were strongly Hsp70 membrane-positive. Irradiated, Hsp70-positive tumor cells are targets for Hsp70-peptide stimulated NK cells. Conclusion An irradiation-inducible, tumor-selective Hsp70 membrane localization provides a target structure for Hsp70-peptide stimulated human NK cells.

Profiling of surface-bound proteins uncovers a tumor-selective heat shock protein 70 (Hsp70) membrane expression that provides a target structure for human NK cells. Hsp70 peptide TKD (TKDNNLLGRFELSG; aa 450–463) was found to enhance the cytolytic activity of NK cells. In this study, we demonstrate that TKD-activated CD3−CD56+CD94+ NK cells are selectively attracted by Hsp70 membrane-positive tumor cells, and supernatants derived thereof. Hsp70 membrane-negative tumors failed to attract these NK cells. The capacity to migrate was associated with a substantial lytic activity against Hsp70-positive tumor cells. Because NK cell migration was independent of cell-to-cell contact, the involvement of a soluble factor was assumed. Interestingly, synthetic Hsp70 protein and Hsp70 peptide TKD, mimicking surface-bound Hsp70, initiates migration of NK cells in a concentration-dependent (1–5 μg/ml), highly selective, and chemokine-independent manner. In summary, our results indicate that Hsp70 peptide TKD not only stimulates cytolysis but also chemotaxis in CD3−CD56+CD94+ NK cells.

Human tumors frequently present heat shock protein 70 (Hsp70) on their cell membranes, whereas corresponding normal tissues fail to do so. Therefore, an Hsp70 membrane-positive phenotype provided a tumor-specific marker. Moreover, membrane-bound Hsp70 provides a target structure for the cytolytic attack mediated by natural killer (NK) cells. Vitamin A derivatives 13-cis retinoic acid (13-RA) and all-trans retinoic acid (ATRA) and sodium-butyrate (SBU) are known for their redifferentiating capacity. Therefore, we asked the question whether loss in tumorigenicity might be associated with a reduced Hsp70 membrane expression. For our studies we used epithelial colon (CX+/CX-) and thyroid (ML-1) cancer cells, with initially different Hsp70 cell surface expression pattern. After treatment up to 7 weeks with freshly prepared 13-RA, ATRA, and SBU at nonlethal concentrations of 10 microM, 1 microM, and 0.5 mM, respectively, growth morphology, Hsp70 levels, and sensitivity toward Hsp70-specific NK cells were compared with that of untreated tumor cells. Significant growth delay was determined in CX+ tumor cells after 6 weeks treatment with 13-RA. Concomitantly, growth morphology changed from spheroid cell clusters to monolayers. Despite a weak increase in cytosolic Hsp70, the percentage of Hsp70 membrane-positive cells dropped significantly after repeated treatments with 13-RA and ATRA in CX+ and ML-1 but not in CX- tumor cells. Similar results were observed with SBU. Functionally, the decrease in Hsp70 membrane-positive CX+ and ML-1 cells correlated with a reduced sensitivity to lysis mediated by NK cells. In summary, redifferentiating agents predominantly affected Hsp70 membrane-positive tumors. The decrease in Hsp70 membrane positivity correlated with a lower sensitivity to NK lysis, growth delay, and altered growth morphology.