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.

Tumor imaging and targeting potential of an Hsp70-derived 14-mer peptide.

BACKGROUND: We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70) on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding 'healthy' tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG), the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+) tumor cells, in vitro. METHODOLOGY/PRINCIPAL FINDINGS: The specificity of carboxy-fluorescein (CF-) labeled TPP (TPP) to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+) and MDA-MB-231 (75% memHsp70+) cells compared to T47D cells (29% memHsp70+) that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the specific binding and rapid internalization of TPP by tumor cells with a memHsp70+ phenotype. TPP might therefore have potential for targeting and imaging the large proportion of tumors (∼50%) that express memHsp70.

Hsp70--a biomarker for tumor detection and monitoring of outcome of radiation therapy in patients with squamous cell carcinoma of the head and neck.

BACKGROUND: Tumor but not normal cells frequently overexpress heat shock protein 70 (Hsp70) and present it on their cell surface (mHsp70) from where it can be actively released. Therefore, membrane (mHsp70) and soluble Hsp70 (sHsp70) were investigated as potential tumor biomarkers and for monitoring the outcome of radiation therapy. METHODS: Biopsies and blood were collected from patients with squamous cell carcinoma of the head and neck (SCCHN) at different time points (before, during therapy and in the follow-up period). Hsp70 membrane expression was determined on single cell suspensions of tumor biopsies and reference tissues by flow cytometry, sHsp70 protein and antibody levels were determined in the serum of patients and healthy donors by ELISA and NK cell markers that are related to the presence of sHsp70 were analyzed in the patient's peripheral blood lymphocytes (PBL). RESULTS: Tumor biopsies exhibited significantly increased mHsp70 expression levels compared to the reference tissue. Soluble Hsp70 levels were significantly higher in SCCHN patients compared to healthy human volunteers and high mHsp70 expression levels on tumor cells were associated with high sHsp70 levels in the serum of patients. Following surgery and radiotherapy sHsp70 levels in patients dropped in patients without tumor relapse in the follow-up period. In contrast to sHsp70 protein, anti-Hsp70 antibody levels remained nearly unaltered in the serum of SCCHN patients before and after therapy. Furthermore, sHsp70 protein but not anti-Hsp70 antibody levels were found to be associated with the tumor volume in SCCHN patients before start of therapy. The expression densities of the activatory NK cell markers CD56, CD94, NKG2D, NKp30, Nkp44, and NKp46 differed in patients following therapeutic intervention. A significant increase in the density of NKG2D was observed in SCCHN patients in the follow-up period after surgery and radiotherapy. CONCLUSION: We suggest sHsp70 as a potential biomarker for detecting tumors and for monitoring the clinical outcome of radiotherapy in SCCHN patients.

Adhesion molecule expression and function of primary endothelial cells in benign and malignant tissues correlates with proliferation.

BACKGROUND: Comparative analysis of the cellular biology of the microvasculature in different tissues requires the availability of viable primary endothelial cells (ECs). This study describes a novel method to isolate primary ECs from healthy organs, repair blastemas and tumors as examples of non-proliferating and proliferating benign and malignant tissues and their functional characterization. METHODOLOGY/PRINCIPAL FINDINGS: Single cell suspensions from hearts, lungs, repair blastemas and tumors were incubated consecutively with an anti-CD31 antibody and magnetic micro-beads, coupled to a derivative of biotin and streptavidin, respectively. Following magnetic bead separation, CD31-positive ECs were released by biotin-streptavidin competition. In the absence of micro-beads, ECs became adherent to plastic surfaces. ECs from proliferating repair blastemas and tumors were larger and exhibited higher expression densities of CD31, CD105 and CD102 compared to those from non-proliferating normal tissues such as heart and lung. The expression density of CD34 was particularly high in tumor-derived ECs, and that of CD54 and CD144 in ECs of repair blastemas. Functionally, ECs of non-proliferating and proliferating tissues differed in their capacity to form tubes in matrigel and to align under flow conditions. CONCLUSIONS/SIGNIFICANCE: This method provides a powerful tool to generate high yields of viable, primary ECs of different origins. The results suggest that an altered expression of adhesion molecules on ECs in proliferating tissues contribute to loss of EC function that might cause a chaotic tumor vasculature.