Elevated circulating Hsp70 levels are correlative for malignancies in different mammalian species.

Circulating Hsp70 levels were determined in feline and porcine cohorts using two different ELISA systems. These comparative animal models of larger organisms often reflect diseases, and especially malignant tumors, better than conventional rodent models. It is therefore essential to investigate the biology and utility of tumor biomarkers in animals such as cats and pigs. In this study, levels of free Hsp70 in the blood of cats with spontaneously occurring tumors were detected using a commercial Hsp70 ELISA (R&D Systems). Sub-analysis of different tumor groups revealed that animals with tumors of epithelial origin presented with significantly elevated circulating Hsp70 concentrations. In addition to free Hsp70 levels measured with the R&D Systems Hsp70 ELISA, levels of exosomal Hsp70 were determined using the compHsp70 ELISA in pigs. Both ELISA systems detected significantly elevated Hsp70 levels (R&D Systems: median 24.9 ng/mL; compHsp70: median 44.2 ng/mL) in the blood of a cohort of APC1311/+ pigs diagnosed with high-grade adenoma polyps, and the R&D Systems Hsp70 ELISA detected also elevated Hsp70 levels in animals with low-grade polyps. In contrast, in flTP53R167H pigs, suffering from malignant osteosarcoma, the compHsp70 ELISA (median 674.32 ng/mL), but not the R&D Systems Hsp70 ELISA (median 4.78 ng/mL), determined significantly elevated Hsp70 concentrations, indicating that in tumor-bearing animals, the dominant form of Hsp70 is of exosomal origin. Our data suggest that both ELISA systems are suitable for detecting free circulating Hsp70 levels in pigs with high-grade adenoma, but only the compHsp70 ELISA can measure elevated, tumor-derived exosomal Hsp70 levels in tumor-bearing animals.

Hsp70-A Universal Biomarker for Predicting Therapeutic Failure in Human Female Cancers and a Target for CTC Isolation in Advanced Cancers.

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.