In contrast to normal cells, tumor cells of multiple entities overexpress the Heat shock protein 70 (Hsp70) not only in the cytosol, but also present it on their plasma membrane in a tumor-specific manner. Furthermore, membrane Hsp70-positive tumor cells actively release Hsp70 in small extracellular vesicles with biophysical characteristics of exosomes. Due to conformational changes of Hsp70 in a lipid environment, most commercially available antibodies fail to detect membrane-bound and vesicular Hsp70. To fill this gap and to assess the role of vesicular Hsp70 in circulation as a potential tumor biomarker, we established the novel complete (comp)Hsp70 sandwich ELISA, using two monoclonal antibodies (mAbs), that is able to recognize both free and lipid-associated Hsp70 on the cell surface of viable tumor cells and on small extracellular vesicles. The epitopes of the mAbs cmHsp70.1 (aa 451-461) and cmHsp70.2 (aa 614-623) that are conserved among different species reside in the substrate-binding domain of Hsp70 with measured affinities of 0.42 nM and 0.44 nM, respectively. Validation of the compHsp70 ELISA revealed a high intra- and inter-assay precision, linearity in a concentration range of 1.56 to 25 ng/mL, high recovery rates of spiked liposomal Hsp70 (>84%), comparable values between human serum and plasma samples and no interference by food intake or age of the donors. Hsp70 concentrations in the circulation of patients with glioblastoma, squamous cell or adeno non-small cell lung carcinoma (NSCLC) at diagnosis were significantly higher than those of healthy donors. Hsp70 concentrations dropped concomitantly with a decrease in viable tumor mass upon irradiation of patients with approximately 20 Gy (range 18-22.5 Gy) and after completion of radiotherapy (60-70 Gy). In summary, the compHsp70 ELISA presented herein provides a sensitive and reliable tool for measuring free and vesicular Hsp70 in liquid biopsies of tumor patients, levels of which can be used as a tumor-specific biomarker, for risk assessment (i.e., differentiation of grade III vs. IV adeno NSCLC) and monitoring of therapeutic outcomes.
Keywords: Hsp70; glioblastoma; liquid biopsy; non-small cell lung carcinoma (NSCLC); prediction; response monitoring; sandwich ELISA; small extracellular vesicles; tumor biomarker.