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

SIMPLE SUMMARY: This study reveals that circulating Hsp70 levels could serve as a tumor biomarker for patients with NSCLC in advanced UICC stages. All patients in advanced tumor stages had significantly elevated Hsp70 levels in the circulation compared to a healthy control cohort and an early-stage tumor cohort, and Hsp70 levels progressively increased with higher UICC tumor stages. These findings demonstrate the potential of Hsp70 measurements to predict an advanced tumor stage in NSCLC patients. We have also demonstrated that the prevalence of CD3−/CD94+ NK cells and CD8+ cytotoxic T cells were greater in advanced tumor stages, whereas that of CD4+ T helper cells was decreased. We hypothesize that raised levels of circulating Hsp70 in higher tumor stages might support NK cell proliferation, but that a lowered prevalence of CD4+ T helper cells could temper the capacity of cytolytic CD8+ T cells and NK cells to control tumor growth. ABSTRACT: 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.