Biomarkers in Adult-Type Diffuse Gliomas: Elevated Levels of Circulating Vesicular Heat Shock Protein 70 Serve as a Biomarker in Grade 4 Glioblastoma and Increase NK Cell Frequencies in Grade 3 Glioma.

The presence of circulating Hsp70 levels and their influence on the immunophenotype of circulating lymphocyte subsets were examined as diagnostic/prognostic biomarkers for the overall survival (OS) in patients with IDH-mutant WHO grade 3 oligodendroglioma, astrocytoma, and IDH-wildtype grade 4 glioblastoma (GBM). Vesicular and free Hsp70 in the plasma/serum was measured using the Hsp70-exo and R&D Systems DuoSet® Hsp70 ELISAs. The immunophenotype and membrane Hsp70 status was determined by multiparameter flow cytometry on peripheral blood lymphocytes and single-cell suspensions of tumor specimens and cultured cells. Compared to healthy controls, circulating vesicular Hsp70 levels were significantly increased in patients with GBM, concomitant with a significant decrease in the proportion of CD3+/CD4+ helper T cells, whereas the frequency of NK cells was most prominently increased in patients with grade 3 gliomas. Elevated circulating Hsp70 levels and a higher prevalence of activated CD3-/CD56+/CD94+/CD69+ NK cells were associated with an improved OS in grade 3 gliomas, whereas high Hsp70 levels and low CD3+/CD4+ frequencies were associated with an adverse OS in GBM. It is assumed that a reduced membrane Hsp70 density on grade 4 versus grade 3 primary glioma cells and reduced CD3+/CD4+ T cell counts in GBM might drive an immunosuppressive tumor microenvironment.

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 in Liquid Biopsies-A Tumor-Specific Biomarker for Detection and Response Monitoring in Cancer.

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.