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.

Immunohistochemical, Flow Cytometric, and ELISA-Based Analyses of Intracellular, Membrane-Expressed, and Extracellular Hsp70 as Cancer Biomarkers.

The major stress-inducible 70 kDa heat shock (stress) protein 70 (Hsp70) is frequently overexpressed in highly aggressive tumor cells and thus might serve as a tumor-specific biomarker of aggressive disease and/or therapeutic resistance. We have previously shown that, in contrast to normal cells, tumor cells present Hsp70 on their plasma membrane. In order to elucidate the role of intracellular, membrane-bound and extracellular Hsp70 as a potential tumor biomarker in cancer, herein we describe protocols for the staining of cytosolic Hsp70 in tumor formalin-fixed paraffin-embedded (FFPE) sections from patients with glioblastoma multiforme using immunohistochemistry, for detecting the expression of plasma membrane-bound Hsp70 by a range of cancer-derived cells using multi-parametric flow cytometry using the cmHsp70.1 monoclonal antibody (mAb) and for the measurement of free and vesicular-associated Hsp70 in the circulation of patients with cancer using a unique enzyme-linked immunosorbent assay (ELISA).

Circulating Hsp70 Levels and the Immunophenotype of Peripheral Blood Lymphocytes as Potential Biomarkers for Advanced Lung Cancer and Therapy Failure after Surgery.

Lung cancer remains a devastating disease with a poor clinical outcome. A biomarker signature which could distinguish lung cancer from metastatic disease and detect therapeutic failure would significantly improve patient management and allow for individualized, risk-adjusted therapeutic decisions. In this study, circulating Hsp70 levels were measured using ELISA, and the immunophenotype of the peripheral blood lymphocytes were measured using multiparameter flow cytometry, to identify a predictive biomarker signature for lung cancer patients pre- and post-operatively, in patients with lung metastases and in patients with COPD as an inflammatory lung disease. The lowest Hsp70 concentrations were found in the healthy controls followed by the patients with advanced COPD. Hsp70 levels sequentially increased with an advancing tumor stage and metastatic disease. In the early-recurrence patients, Hsp70 levels started to increase within the first three months after surgery, but remained unaltered in the recurrence-free patients. An early recurrence was associated with a significant drop in B cells and an increase in Tregs, whereas the recurrence-free patients had elevated T and NK cell levels. We conclude that circulating Hsp70 concentrations might have the potential to distinguish lung cancer from metastatic disease, and might be able to predict an advanced tumor stage and early recurrence in lung cancer patients. Further studies with larger patient cohorts and longer follow-up periods are needed to validate Hsp70 and immunophenotypic profiles as predictive biomarker signatures.

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.

Elevated Levels of Circulating Hsp70 and an Increased Prevalence of CD94+/CD69+ NK Cells Is Predictive for Advanced Stage Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the second most frequently diagnosed tumor worldwide. Despite the clinical progress which has been achieved by multimodal therapies, including radiochemotherapy, and immune checkpoint inhibitor blockade, the overall survival of patients with advanced-stage NSCLC remains poor, with less than 16 months. It is well established that many aggressive tumor entities, including NSCLC, overexpress the major stress-inducible heat shock protein 70 (Hsp70) in the cytosol, present it on the plasma membrane in a tumor-specific manner, and release Hsp70 into circulation. Although high Hsp70 levels are associated with tumor aggressiveness and therapy resistance, membrane-bound Hsp70 can serve as a tumor-specific antigen for Hsp70-primed natural killer (NK) cells, expressing the C-type lectin receptor CD94, which is part of the activator receptor complex CD94/NKG2C. Therefore, we investigated circulating Hsp70 levels and changes in the composition of peripheral blood lymphocyte subsets as potential biomarkers for the advanced Union for International Cancer Control (UICC) stages in NSCLC. As expected, circulating Hsp70 levels were significantly higher in NSCLC patients compared to the healthy controls, as well as in patients with advanced UICC stages compared to those in UICC stage I. Smoking status did not influence the circulating Hsp70 levels significantly. Concomitantly, the proportions of CD4+ T helper cells were lower compared to the healthy controls and stage I tumor patients, whereas that of CD8+ cytotoxic T cells was progressively higher. The prevalence of CD3-/CD56+, CD3-/NKp30, CD3-/NKp46+, and CD3-/NKG2D+ NK cells was higher in stage IV/IIIB of the disease than in stage IIIA but were not statistically different from that in healthy individuals. However, the proportion of NK cells expressing CD94 and the activation/exhaustion marker CD69 significantly increased in higher tumor stages compared with stage I and the healthy controls. We speculate that although elevated circulating Hsp70 levels might promote the prevalence of CD94+ NK cells in patients with advanced-stage NSCLC, the cytolytic activity of these NK cells also failed to control tumor growth due to insufficient support by pro-inflammatory cytokines from CD4+ T helper cells. This hypothesis is supported by a comparative multiplex cytokine analysis of the blood in lung cancer patients with a low proportion of CD4+ T cells, a high proportion of NK cells, and high Hsp70 levels versus patients with a high proportion of CD4+ T cells exhibiting lower IL-2, IL-4, IL-6, IFN-γ, granzyme B levels.