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.

BackgroundTumor but not normal cells frequently overexpress heat shock protein 70 (Hsp70) and present it on their cell surface (mHsp70) from where it can be actively released. Therefore, membrane (mHsp70) and soluble Hsp70 (sHsp70) were investigated as potential tumor biomarkers and for monitoring the outcome of radiation therapy.MethodsBiopsies and blood were collected from patients with squamous cell carcinoma of the head and neck (SCCHN) at different time points (before, during therapy and in the follow-up period). Hsp70 membrane expression was determined on single cell suspensions of tumor biopsies and reference tissues by flow cytometry, sHsp70 protein and antibody levels were determined in the serum of patients and healthy donors by ELISA and NK cell markers that are related to the presence of sHsp70 were analyzed in the patient’s peripheral blood lymphocytes (PBL).ResultsTumor biopsies exhibited significantly increased mHsp70 expression levels compared to the reference tissue. Soluble Hsp70 levels were significantly higher in SCCHN patients compared to healthy human volunteers and high mHsp70 expression levels on tumor cells were associated with high sHsp70 levels in the serum of patients. Following surgery and radiotherapy sHsp70 levels in patients dropped in patients without tumor relapse in the follow-up period. In contrast to sHsp70 protein, anti-Hsp70 antibody levels remained nearly unaltered in the serum of SCCHN patients before and after therapy. Furthermore, sHsp70 protein but not anti-Hsp70 antibody levels were found to be associated with the tumor volume in SCCHN patients before start of therapy. The expression densities of the activatory NK cell markers CD56, CD94, NKG2D, NKp30, Nkp44, and NKp46 differed in patients following therapeutic intervention. A significant increase in the density of NKG2D was observed in SCCHN patients in the follow-up period after surgery and radiotherapy.ConclusionWe suggest sHsp70 as a potential biomarker for detecting tumors and for monitoring the clinical outcome of radiotherapy in SCCHN patients.

BackgroundWe have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70) on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding ’healthy‘ tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG), the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+) tumor cells, in vitro.Methodology/Principal FindingsThe specificity of carboxy-fluorescein (CF-) labeled TPP (TPP) to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+) and MDA-MB-231 (75% memHsp70+) cells compared to T47D cells (29% memHsp70+) that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization.Conclusions/SignificanceThis study demonstrates the specific binding and rapid internalization of TPP by tumor cells with a memHsp70+ phenotype. TPP might therefore have potential for targeting and imaging the large proportion of tumors (∼50%) that express memHsp70.