RESUMO
Heat shock protein 70 (Hsp70) is frequently overexpressed in many different tumor types. However, Hsp70 has also been shown to be selectively presented on the plasma membrane of tumor cells, but not normal cells, and this membrane form of Hsp70 (mHsp70) could be considered a universal tumor biomarker. Since viable, mHsp70-positive tumor cells actively release Hsp70 in lipid micro-vesicles, we investigated the utility of Hsp70 in circulation as a universal tumor biomarker and its potential as an early predictive marker of therapeutic failure. We have also evaluated mHsp70 as a target for the isolation and enumeration of circulating tumor cells (CTCs) in patients with different tumor entities. Circulating vesicular Hsp70 levels were measured in the peripheral blood of tumor patients with the compHsp70 ELISA. CTCs were isolated using cmHsp70.1 and EpCAM monoclonal antibody (mAb)-based bead approaches and characterized by immunohistochemistry using cytokeratin and CD45-specific antibodies. In two out of 35 patients exhibiting therapeutic failure two years after initial diagnosis of non-metastatic breast cancer, progressively increasing levels of circulating Hsp70 had already been observed during therapy, whereas levels in patients without subsequent recurrence remained unaltered. With regards to CTC isolation from patients with different tumors, an Hsp70 mAb-based selection system appears superior to an EpCAM mAb-based approach. Extracellular and mHsp70 can therefore serve as a predictive biomarker for therapeutic failure in early-stage tumors and as a target for the isolation of CTCs in various tumor diseases.
RESUMO
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.
