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.

Targeting Cancer Metabolism Breaks Radioresistance by Impairing the Stress Response.

The heightened energetic demand increases lactate dehydrogenase (LDH) activity, the corresponding oncometabolite lactate, expression of heat shock proteins (HSPs) and thereby promotes therapy resistance in many malignant tumor cell types. Therefore, we assessed the coregulation of LDH and the heat shock response with respect to radiation resistance in different tumor cells (B16F10 murine melanoma and LS174T human colorectal adenocarcinoma). The inhibition of LDH activity by oxamate or GNE-140, glucose deprivation and LDHA/B double knockout (LDH-/-) in B16F10 and LS174T cells significantly diminish tumor growth; ROS production and the cytosolic expression of different HSPs, including Hsp90, Hsp70 and Hsp27 concomitant with a reduction of heat shock factor 1 (HSF1)/pHSF1. An altered lipid metabolism mediated by a LDHA/B double knockout results in a decreased presence of the Hsp70-anchoring glycosphingolipid Gb3 on the cell surface of tumor cells, which, in turn, reduces the membrane Hsp70 density and increases the extracellular Hsp70 levels. Vice versa, elevated extracellular lactate/pyruvate concentrations increase the membrane Hsp70 expression in wildtype tumor cells. Functionally, an inhibition of LDH causes a generalized reduction of cytosolic and membrane-bound HSPs in tumor cells and significantly increases the radiosensitivity, which is associated with a G2/M arrest. We demonstrate that targeting of the lactate/pyruvate metabolism breaks the radioresistance by impairing the stress response.

Potential Role of Hsp70 and Activated NK Cells for Prediction of Prognosis in Glioblastoma Patients.

Despite rapid progress in the treatment of many cancers, glioblastoma remains a devastating disease with dismal prognosis. The aim of this study was to identify chaperone- and immune-related biomarkers to improve prediction of outcome in glioblastoma. Depending on its intra- or extracellular localization the major stress-inducible heat shock protein 70 (Hsp70) fulfills different tasks. In the cytosol Hsp70 interferes with pro-apoptotic signaling pathways and thereby protects tumor cells from programmed cell death. Extracellular Hsp70 together with pro-inflammatory cytokines are reported to stimulate the expression of activatory NK cell receptors, recognizing highly aggressive human tumor cells that present Hsp70 on their cell surface. Therefore, intra-, extracellular and membrane-bound Hsp70 levels were assessed in gliomas together with activatory NK cell receptors. All gliomas were found to be membrane Hsp70-positive and high grade gliomas more frequently show an overexpression of Hsp70 in the nucleus and cytosol. Significantly elevated extracellular Hsp70 levels are detected in glioblastomas with large necrotic areas. Overall survival (OS) is more favorable in patients with low Hsp70 serum levels indicating that a high Hsp70 expression is associated with an unfavorable prognosis. The data provide a first hint that elevated frequencies of activated NK cells at diagnosis might be associated with a better clinical outcome.

Targeted Natural Killer Cell-Based Adoptive Immunotherapy for the Treatment of Patients with NSCLC after Radiochemotherapy: A Randomized Phase II Clinical Trial.

PURPOSE: Non-small cell lung cancer (NSCLC) is a fatal disease with poor prognosis. A membrane-bound form of Hsp70 (mHsp70) which is selectively expressed on high-risk tumors serves as a target for mHsp70-targeting natural killer (NK) cells. Patients with advanced mHsp70-positive NSCLC may therefore benefit from a therapeutic intervention involving mHsp70-targeting NK cells. The randomized phase II clinical trial (EudraCT2008-002130-30) explores tolerability and efficacy of ex vivo-activated NK cells in patients with NSCLC after radiochemotherapy (RCT). PATIENTS AND METHODS: Patients with unresectable, mHsp70-positive NSCLC (stage IIIa/b) received 4 cycles of autologous NK cells activated ex vivo with TKD/IL2 [interventional arm (INT)] after RCT (60-70 Gy, platinum-based chemotherapy) or RCT alone [control arm (CTRL)]. The primary objective was progression-free survival (PFS), and secondary objectives were the assessment of quality of life (QoL, QLQ-LC13), toxicity, and immunobiological responses. RESULTS: The NK-cell therapy after RCT was well tolerated, and no differences in QoL parameters between the two study arms were detected. Estimated 1-year probabilities for PFS were 67% [95% confidence interval (CI), 19%-90%] for the INT arm and 33% (95% CI, 5%-68%) for the CTRL arm (P = 0.36, 1-sided log-rank test). Clinical responses in the INT group were associated with an increase in the prevalence of activated NK cells in their peripheral blood. CONCLUSIONS: Ex vivo TKD/IL2-activated, autologous NK cells are well tolerated and deliver positive clinical responses in patients with advanced NSCLC after RCT.

Gold Nanoparticle Mediated Multi-Modal CT Imaging of Hsp70 Membrane-Positive Tumors.

Imaging techniques such as computed tomographies (CT) play a major role in clinical imaging and diagnosis of malignant lesions. In recent years, metal nanoparticle platforms enabled effective payload delivery for several imaging techniques. Due to the possibility of surface modification, metal nanoparticles are predestined to facilitate molecular tumor targeting. In this work, we demonstrate the feasibility of anti-plasma membrane Heat shock protein 70 (Hsp70) antibody functionalized gold nanoparticles (cmHsp70.1-AuNPs) for tumor-specific multimodal imaging. Membrane-associated Hsp70 is exclusively presented on the plasma membrane of malignant cells of multiple tumor entities but not on corresponding normal cells, predestining this target for a tumor-selective in vivo imaging. In vitro microscopic analysis revealed the presence of cmHsp70.1-AuNPs in the cytosol of tumor cell lines after internalization via the endo-lysosomal pathway. In preclinical models, the biodistribution as well as the intratumoral enrichment of AuNPs were examined 24 h after i.v. injection in tumor-bearing mice. In parallel to spectral CT analysis, histological analysis confirmed the presence of AuNPs within tumor cells. In contrast to control AuNPs, a significant enrichment of cmHsp70.1-AuNPs has been detected selectively inside tumor cells in different tumor mouse models. Furthermore, a machine-learning approach was developed to analyze AuNP accumulations in tumor tissues and organs. In summary, utilizing mHsp70 on tumor cells as a target for the guidance of cmHsp70.1-AuNPs facilitates an enrichment and uniform distribution of nanoparticles in mHsp70-expressing tumor cells that enables various microscopic imaging techniques and spectral-CT-based tumor delineation in vivo.

Time- and Dose-Dependent Effects of Ionizing Irradiation on the Membrane Expression of Hsp70 on Glioma Cells.

The major stress-inducible protein Hsp70 (HSPA1A) is overexpressed in the cytosol of many highly aggressive tumor cells including glioblastoma multiforme and presented on their plasma membrane. Depending on its intracellular or membrane localization, Hsp70 either promotes tumor growth or serves as a target for natural killer (NK) cells. The kinetics of the membrane Hsp70 (mHsp70) density on human glioma cells (U87) was studied after different irradiation doses to define the optimal therapeutic window for Hsp70-targeting NK cells. To maintain the cells in the exponential growth phase during a cultivation period of 7 days, different initial cell counts were seeded. Although cytosolic Hsp70 levels remained unchanged on days 4 and 7 after a sublethal irradiation with 2, 4 and 6 Gy, a dose of 2 Gy resulted in an upregulated mHsp70 density in U87 cells which peaked on day 4 and started to decline on day 7. Higher radiation doses (4 Gy, 6 Gy) resulted in an earlier and more rapid onset of the mHsp70 expression on days 2 and 1, respectively, followed by a decline on day 5. Membrane Hsp70 levels were higher on cells in G2/M than in G1; however, an irradiation-induced cell cycle arrest on days 4 and 7 was not associated with an increase in the mHsp70 density. Extracellular Hsp70 concentrations in the supernatant of irradiated cells were significantly higher than sham (0 Gy) irradiated cells on days 4 and 7, but not on day 1. Functionally, elevated mHsp70 densities were associated with a significantly better lysis by Hsp70-targeting NK cells. In summary, the kinetics of changes in the mHsp70 density upon irradiation on tumor cells is time- and dose-dependent.

Increased heat shock protein 70 (Hsp70) serum levels and low NK cell counts after radiotherapy - potential markers for predicting breast cancer recurrence?

BACKGROUND: Breast cancer is the most common invasive tumor in women worldwide and the second cause of cancer-related deaths. After breast conserving surgery the tumor bed gets irradiated. Radiation-induced tumor cell death has been found to be associated with the release of damage-associated molecular patterns (DAMPs) including free Hsp70 that can stimulate inflammatory immune responses. Therefore, Hsp70 serum levels as well as the composition of lymphocyte subpopulations have been measured in breast cancer patients during therapy and in the follow-up period as potential predictors for clinical outcome. METHODS: The serum of 40 breast cancer patients, who received a breast-conserving surgery and adjuvant radiotherapy (RT) was examined for soluble, free Hsp70 using the R&D Human HSP70 DuoSet and lipHsp70 ELISA. Lymphocyte subpopulations and total lymphocyte counts were analysed by multiparameter flow cytometry in the peripheral blood. Blood samples were collected before (t1), after 30 Gy (t2) and 60 Gy (t3), 6 weeks (t4), 6 months (t5) and 1 year (t6) after RT. Clinical responses were assessed regularly up to 5 years after RT. RESULTS: Patients who developed a contralateral recurrence or metastases within the first 2 years after RT had significantly higher serum Hsp70 values at the end of RT (t3; p = 0.03) up to 6 weeks after RT (t4; p = 0.007) compared to patients who either remained disease-free or developed a secondary endometrial carcinoma. Clinicopathological parameters such as age, tumor size, grading and TNM-stage of the resected tumors, adjuvant chemotherapy and irradiation dose did not affect serum Hsp70 levels. Elevated free Hsp70 levels might be indicative for a chronic inflammatory response which could support tumor recurrence. Lymphocyte subpopulation analysis revealed lower NK cell counts after RT in recurrence/metastases patients as compared to disease-free patients. In contrast, no significant changes were observed in the proportion of T and B cells. CONCLUSION: Longitudinal elevated serum levels of free Hsp70 up to 6 weeks after RT and dropping NK cell counts might be predictive for an unfavourable prognosis in patients with breast cancer.

Membrane Hsp70-A Novel Target for the Isolation of Circulating Tumor Cells After Epithelial-to-Mesenchymal Transition.

The presence of circulating tumor cells (CTCs) in the peripheral blood is a pre-requisite for progression, invasion, and metastatic spread of cancer. Consequently, the enumeration and molecular characterization of CTCs from the peripheral blood of patients with solid tumors before, during and after treatment serves as a valuable tool for categorizing disease, evaluating prognosis and for predicting and monitoring therapeutic responsiveness. Many of the techniques for isolating CTCs are based on the expression of epithelial cell surface adhesion molecule (EpCAM, CD326) on tumor cells. However, the transition of adherent epithelial cells to migratory mesenchymal cells (epithelial-to-mesenchymal transition, EMT)-an essential element of the metastatic process-is frequently associated with a loss of expression of epithelial cell markers, including EpCAM. A highly relevant proportion of mesenchymal CTCs cannot therefore be isolated using techniques that are based on the "capture" of cells expressing EpCAM. Herein, we provide evidence that a monoclonal antibody (mAb) directed against a membrane-bound form of Hsp70 (mHsp70)-cmHsp70.1-can be used for the isolation of viable CTCs from peripheral blood of tumor patients of different entities in a more quantitative manner. In contrast to EpCAM, the expression of mHsp70 remains stably upregulated on migratory, mesenchymal CTCs, metastases and cells that have been triggered to undergo EMT. Therefore, we propose that approaches for isolating CTCs based on the capture of cells that express mHsp70 using the cmHsp70.1 mAb are superior to those based on EpCAM expression.

[Benefits of meditation on the hypertensive adult]. / Bénéfices de la meditation chez l'adulte hypertendu.

The prevalence of high blood pressure is increasing and expanding preventive and treatment measures may be useful. Some data in the literature suggest that meditation can play a role not only in stress management but also in blood pressure control and cardiovascular disease prevention. Mechanisms involved may be based on the regulation of emotional and biological responses to stress, by regulation of the autonomous nervous system. This approach is available to everyone interested, has no adverse effects and is not related to any particular religious beliefs.

La prévalence de l'hypertension artérielle est en augmentation et élargir le spectre des mesures à disposition pour sa prévention et son traitement peut être utile. Certaines données de la littérature suggèrent que la méditation pourrait jouer un rôle non seulement dans la gestion du stress, mais aussi dans le contrôle tensionnel et la prévention des maladies cardiovasculaires. Les mécanismes impliqués reposeraient sur la modulation de la réaction émotionnelle et la réponse biologique au stress, via la régulation du système nerveux autonome. Il s'agit d'une méthode accessible à toute personne intéressée, dénuée d'effets secondaires et ne reposant pas sur un système de croyance particulier.

Donor insemination and parenting: concerns and strategies of lesbian couples. A review of international studies.

The concerns and strategies of lesbian couples at the time of donor insemination, throughout pregnancy and during early parenthood differ in several ways from those of heterosexual couples. For the majority of the lesbian couples insemination was the first option to conceive. They reported having strategies for their children growing up in a heteronormative society, and the parents shared time with and responsibility for the children equally. Issues considered most important by the couple were an open and non-judgmental attitude from caregivers without heteronormative assumptions, knowledge about lesbian women's situation and acknowledgement of the co-mother.