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

Abstract

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.

HSP70 Multi-Functionality in Cancer

“…Interestingly, in contrast to EpCAM, the EMT does not downregulate HSP70 expression as higher membrane-bound HSP70 (mHSP70) expression has been found on metastatic tumors compared to its primary counterparts [131,[206][207][208][209]. In this regard, Stangl and colleagues used HSP70-TKD peptide to develop monoclonal antibody directed against mHSP70 for specific targeting mHSP70-positive tumors by NK cells and efficient isolation of CTCs [206,210]. Taken together, on the one hand, high surface expression of HSP70 is found in metastatic tumors and mHSP70 may serve as a biomarker for assessing the metastatic spread.…”

“…Clinically, circulating tumor cells (CTCs) that underwent EMT serve as a prognostic marker of a metastatic spread and can be isolated from patient's blood using epithelial cell adhesion molecule (EpCAM) as a cell surface marker [206]. Interestingly, in contrast to EpCAM, the EMT does not downregulate HSP70 expression as higher membrane-bound HSP70 (mHSP70) expression has been found on metastatic tumors compared to its primary counterparts [131,[206][207][208][209]. In this regard, Stangl and colleagues used HSP70-TKD peptide to develop monoclonal antibody directed against mHSP70 for specific targeting mHSP70-positive tumors by NK cells and efficient isolation of CTCs [206,210].…”

Molecular Chaperones in Cancer Stem Cells: Determinants of Stemness and Potential Targets for Antitumor Therapy

“…Later, plasma membrane-bound HSP70 was proposed to be used as a specific easily detectable marker of CSC-like circulating tumor cells that have undergone EMT and consequently lost the epithelial cell surface markers EpCAM and CD326 [126]. As well as HSP90, HSP70 was found in exosomes secreted by prostate cancer cells undergoing hypoxic stress [20].…”

Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A

“…Many reports revealed that HspA1A is found in association with sphingomyelin/cholesterol rich lipid domains (lipid rafts) [ 5 , 18 , 19 , 20 , 21 ]. Therefore, several investigators aim to target the cell surface/PM-localized HspA1A as a novel anti-cancer treatment [ 5 , 22 , 23 , 24 ]. It is also fairly well established that HspA1A’s PM localization depends on its interaction with intracellular PS and other lipids, including globotriaoslyceramide, which is primarily found in lipid rafts [ 6 , 7 , 25 ].…”