Heat shock protein 70 (Hsp70) is frequently overexpressed in many different tumor types. However, Hsp70 has also been shown to be selectively presented on the plasma membrane of tumor cells, but not normal cells, and this membrane form of Hsp70 (mHsp70) could be considered a universal tumor biomarker. Since viable, mHsp70-positive tumor cells actively release Hsp70 in lipid micro-vesicles, we investigated the utility of Hsp70 in circulation as a universal tumor biomarker and its potential as an early predictive marker of therapeutic failure. We have also evaluated mHsp70 as a target for the isolation and enumeration of circulating tumor cells (CTCs) in patients with different tumor entities. Circulating vesicular Hsp70 levels were measured in the peripheral blood of tumor patients with the compHsp70 ELISA. CTCs were isolated using cmHsp70.1 and EpCAM monoclonal antibody (mAb)-based bead approaches and characterized by immunohistochemistry using cytokeratin and CD45-specific antibodies. In two out of 35 patients exhibiting therapeutic failure two years after initial diagnosis of non-metastatic breast cancer, progressively increasing levels of circulating Hsp70 had already been observed during therapy, whereas levels in patients without subsequent recurrence remained unaltered. With regards to CTC isolation from patients with different tumors, an Hsp70 mAb-based selection system appears superior to an EpCAM mAb-based approach. Extracellular and mHsp70 can therefore serve as a predictive biomarker for therapeutic failure in early-stage tumors and as a target for the isolation of CTCs in various tumor diseases.

Stress-inducible heat shock protein 70 (Hsp70), which functions as a molecular chaperone and is frequently overexpressed in different cancer cell types, is present on the cell surface of tumor cells and is actively released into the circulation in free and extracellular lipid vesicle-associated forms. Since the exact pathomechanism of endometriosis has not yet been elucidated (although it has been associated with the development of endometrial and ovarian cancer), we asked whether extracellular Hsp70 and circulating endometriotic cells (CECs) reflect the presence and development of endometriosis. Therefore, circulating levels of free and lipid microvesicle-associated Hsp70 were measured using the Hsp70-exo ELISA, and the presence of circulating CECs in the peripheral blood of patients with endometriosis was determined using membrane Hsp70 (mHsp70) and EpCAM monoclonal antibody (mAb)-based bead isolation approaches. Isolated CECs were further characterized by immunofluorescence using reagents directed against cytokeratin (epithelial marker), CD45 (leukocyte marker), CD105/CD44 (mesenchymal stemness markers) and by comparative RNA analysis. Similar to the situation in patients with cancer, the levels of circulating Hsp70 were elevated in the blood of patients with histologically proven endometriosis compared to a healthy control cohort, with significantly elevated Hsp70 levels in endometriosis patients with lesions outside the uterine cavity. Moreover, CECs could be isolated using the cmHsp70.1 mAb-based, and to a lesser extent EpCAM mAb-based, bead approach in all patients with endometriosis, with the highest counts obtained using the mHsp70-targeting procedure in patients with extra-uterine involvement. The longevity in cell culture and the expression of the cytokeratins CD105 and CD44, together with differentially expressed genes related to epithelial-to-mesenchymal transition (EMT), revealed similarities between mHsp70-expressing CECs and circulating tumor cells (CTCs) and suggest a mesenchymal stem cell origin. These findings support the involvement of mHsp70-positive stem cell-like cells in the development of endometriotic lesions. In summary, elevated levels of Hsp70 and CECs in the circulation could serve as liquid biopsy markers for endometriosis with extra-uterine involvement and help to elucidate the underlying pathomechanism of the disease.

The stress-inducible heat shock protein 70 (Hsp70), which functions as a molecular chaperone and is frequently overexpressed in different cancer cell types, is presented on the cell surface of tumor cells and is actively released into the circulation in free and extracellular lipid vesicle-associated forms. Since the exact pathomechanism of endometriosis has not yet been elucidated but has been associated with the development of endometrial and ovarian cancer, we asked whether extracellular Hsp70 and circulating endometriotic cells (CECs) reflects the presence and development of endometriosis. Therefore, circulating levels of free and lipid microvesicle-associated Hsp70 were measured using the Hsp70-exo ELISA, and the presence of circulating CECs in the peripheral blood of patients with endometriosis was determined using membrane Hsp70 (mHsp70) and EpCAM monoclonal antibody (mAb)-based bead isolation approaches. Isolated CECs were further characterized by immunofluorescence using reagents directed against cytokeratin (epithelial), CD45 (leukocyte), CD105/CD44 (mesenchymal-stemness) and by comparative RNA analysis. Similar to the situation in patients with cancer, levels of circulating Hsp70 were elevated in the blood of patients with histologically proven endometriosis compared to a healthy control cohort, with the highest Hsp70 level in a patient with lymph node endometriosis. Moreover, CECs could be isolated using the cmHsp70.1 mAb- and to a lesser extent EpCAM mAb-based bead approaches in all patients with endometriosis. The longevity in cell culture and the expression of cytokeratin, CD105 and CD44, together with differentially expressed genes related to EMT reveals similarities of mHsp70 expressing CECs with circulating tumor cells (CTCs) and suggest a mesenchymal-stemness origin. These findings may support the involvement of mHsp70 positive, stem cell-like cells in the development of endometriotic lesions. In summary, elevated levels of Hsp70 in the blood and CECs could serve as liquid biopsy markers for endometriosis and help to elucidate the underlying pathomechanism of the disease.

Background: Liquid biopsy-based biomarkers provide valuable insights into tumor biology, dynamics, burden, relapse prediction and therapeutic responsiveness. The stress-inducible heat shock protein 70 (Hsp70), which is frequently overexpressed in highly aggressive solid tumors and is presented on the cell membrane of tumors but not normal cells, is found in the circulation either as a free protein originating from dying cells or in the context of extracellular vesicles (EVs) that are actively released by viable tumor cells. This study demonstrates the potential value of circulating Hsp70 (eHsp70) levels across multiple solid tumor entities as an entity- and stage-dependent diagnostic biomarker reflecting tumor burden and disease stage. Methods: Circulating eHsp70 levels, as determined using the Hsp70-exo ELISA which detects free and EV-associated Hsp70, in plasma samples collected from patients with different tumor entities (n = 389) prior to the initiation of any oncological therapy and healthy controls (n = 108) between 2021 and 2025, were analyzed retrospectively. Tumor stages were categorized as early, locally advanced, or metastatic. The Kruskal–Wallis test was used for group comparisons and the Receiver Operating Characteristic (ROC) curve was used to evaluate the diagnostic performance of eHsp70 levels. DeLong’s test was used to calculate differences between AUC values. Results: In tumor patients (n = 389), circulating eHsp70 levels were significantly higher than those in healthy controls (n = 108) (Kruskal–Wallis, p < 0.001). eHsp70 levels progressively increased from early-stage to locally advanced and metastatic disease in a stage-dependent manner. Although ROC analysis demonstrated the limited discriminatory performance of eHsp70 levels in early-stage disease (AUC 0.569), increased discrimination was apparent in locally advanced disease (AUC 0.751), metastatic tumors (AUC 0.784) and combined advanced tumor diseases (AUC 0.765; significant by DeLong’s Test comparing early-stage to locally advanced and metastatic tumors), irrespective of the tumor entity with the highest AUC values in metastatic breast cancer (AUC 0.872), sarcoma (AUC 0.861) and non-small cell lung cancer (NSCLC) (AUC 0.835). Apart from minor entity-specific differences, the correlation of eHsp70 levels with the tumor stage remained consistent across all measured tumor entities. Conclusions: Circulating eHsp70 levels are markedly elevated in patients with highly malignant solid tumors and show a consistent, stage-dependent increase across multiple tumor types. These findings suggest that circulating eHsp70, as an indicator of tumor-associated cellular stress and overall tumor burden, represents a valuable biomarker for assessing disease stage, monitoring disease progression, and evaluating therapeutic responses.

Background: Head and neck squamous cell carcinomas (HNSCCs) are highly immunosuppressive, and tumor-associated macrophages constitute a major component of HNSCC tumor microenvironments. Here, we investigate the effects of circulating small extracellular vesicles (sEVs) from HNSCC patients on primary macrophages, to elucidate systemic sEV-mediated immune regulation in HNSCC patients. Methods: sEVs were isolated from plasma by size-exclusion chromatography. Internalization of PKH26-labeled sEVs was demonstrated, and mass spectrometry analysis was performed on HNSCC sEVs and sEV-treated macrophages. NF-κB activation was investigated by Western blot and p65 translocation assay. Downstream, mRNA and protein levels of cytokines and chemotaxis of T cells were investigated. Results: Proteomic analysis revealed time-dependent modulation of the macrophage proteome and NF-κB signaling pathway, which was confirmed by Western blot and p65 translocation assay. sEVs from HNSCC patients negative for human papillomavirus type 16 (HPV) differentially modulated NF-κB signaling and sEV uptake mechanisms compared with sEVs from HPV-positive patients and healthy donors. Conclusions: Circulating sEVs derived from HNSCC patient plasma modulate macrophage proteomic profiles and NF-κB signaling. HPV16 status was associated with differential sEV-mediated macrophage regulation, suggesting a potential role of sEVs in systemic immune modulation in HNSCC.