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Elevated levels of the stress-inducible heat shock protein 70 (Hsp70) in the peripheral circulation have been reported for many tumor entities. In line with these findings we have shown that Hsp70 membrane-positive tumor cells actively release Hsp70 in exosome-like lipid vesicles. Since most commercial Hsp70 Enzyme-Linked Immunosorbent Assays (ELISAs) are not validated for the detection of liposomal Hsp70 in serum, the lipHsp70 ELISA was established using the monoclonal antibody cmHsp70.1 as a detection reagent. This antibody has been reported to recognize free and membrane-bound Hsp70 on living tumor cells. Validation of the ELISA showed a high assay precision and linearity in a concentration range of 0.36-17.4 ng/ml. A comparison of the recovery of spiked Hsp70 in buffer and serum samples revealed a significantly better recovery using the lipHsp70 ELISA compared to a commercial ELISA. With respect to lipid-associated Hsp70 a tenfold higher recovery was found with the lipHsp70 ELISA compared to the commercial ELISA. The analysis of blood samples of healthy human volunteers (n=114) revealed a mean serum Hsp70 concentration of 6.4 ± 2.7 and 2.8 ± 1.3 ng/ml, respectively, using the lipHsp70 and the control ELISA. No significant age-related differences in Hsp70 serum levels were detected. The lipHsp70 ELISA is equally suitable for serum and plasma and the measured Hsp70 concentrations were not impacted by food intake, repeated freezing and thawing of the sample or moderate hemolysis. A comparison of the Hsp70 levels in patients with head and neck, lung, colorectal, pancreatic cancer, glioblastoma or hematological malignancies and healthy human volunteers revealed significantly higher levels in tumor patients. In summary, the lipHsp70 ELISA provides a highly sensitive and robust method for measuring liposomal and free Hsp70 in serum and plasma and thus could provide a useful tool for tumor detection and for monitoring the clinical outcome of patients.

Members of the heat shock protein 70 (HSP70) family play an important role in assisting protein folding, preventing protein aggregation and transport of proteins across membranes under physiological conditions. Following environmental (i.e., irradiation, chemotherapy), physiological (i.e., cell growth, differentiation), and pathophysiological (i.e., inflammation, tumorigenesis) stress, the synthesis of heat shock proteins (HSPs) is highly up-regulated, whereas protein synthesis in general is reduced. In contrast to normal cells, many tumor entities including hepatocellular carcinoma (HCC) overexpress HSP70, the major-stress-inducible member of the HSP70 family, present it on their cell surface and secrete it into the extracellular milieu. Herein, the prognostic relevance of serum HSP70 levels in patients with chronic hepatitis (CH; n = 50), liver cirrhosis (LC; n = 46), and HCC (n = 47) was analyzed. Similar to other tumor entities, HSP70 is also present on the surface of primary HCC cells. The staining intensity of intracellular HSP70 in HCC tissue is stronger compared to control and cirrhotic liver sections. HSP70 serum levels in all HCC patients were significantly higher compared to a control group without liver disease (n = 40). No significant age- and gender-related differences in HSP70 serum levels were observed in male and female healthy human volunteers (n = 86). Patients with CH (n = 50) revealed significantly higher HSP70 serum levels compared to the control group, however, these values were significantly lower than those of HCC patients (n = 47). Furthermore, a subgroup of patients with LC who subsequently developed HCC (LC-HCC, n = 13) revealed higher HSP70 serum levels than patients with LC (n = 46, p = 0.05). These data indicate that serum HSP70 levels are consecutively increased in patients with CH, LC and liver carcinomas and thus might have a prognostic value.