Ectodomain cleavage of cell-surface proteins by A disintegrin and metalloproteinases (ADAMs) is highly regulated, and its dysregulation has been linked to many diseases. ADAM10 and ADAM17 cleave most disease-relevant substrates. Broad-spectrum metalloprotease inhibitors have failed clinically, and targeting the cleavage of a specific substrate has remained impossible. It is therefore necessary to identify signaling intermediates that determine substrate specificity of cleavage. We show here that phorbol ester or angiotensin II-induced proteolytic release of EGF family members may not require a significant increase in ADAM17 protease activity. Rather, inducers activate a signaling pathway using PKC-α and the PKC-regulated protein phosphatase 1 inhibitor 14D that is required for ADAM17 cleavage of TGF-α, heparin-binding EGF, and amphiregulin. A second pathway involving PKC-δ is required for neuregulin (NRG) cleavage, and, indeed, PKC-δ phosphorylation of serine 286 in the NRG cytosolic domain is essential for induced NRG cleavage. Thus, signaling-mediated substrate selection is clearly distinct from regulation of enzyme activity, an important mechanism that offers itself for application in disease.epidermal growth factor receptor | transactivation T he ectodomains of many cell surface proteins are shed from the surface (i.e., "ectodomain shedding") by metalloproteases. Ectodomain shedding generates many diverse bioactive cytokines and growth factors, and governs important cellular processes in the developing and adult organism, including the control of growth, adhesion, and motility of cells (reviewed in refs. 1-3). EGF receptor activation generates signals for cell proliferation, migration, differentiation, or survival. The 12 EGF family members are synthesized as cell surface transmembrane precursors. The active growth factors are released by A disintegrin and metalloproteinases (ADAMs) and activate specific heterodimeric EGF receptors on the cell surface connected to diverse intracellular signaling pathways (4, 5). Increased shedding of EGF ligands has been linked to different clinical pathologic processes (6-10); hence, therapeutic control of ligand release would be beneficial. Of the 12 functional ADAMs encoded in the human genome (3) only two-ADAM10 and ADAM17-handle most of the numerous ADAM substrates, in particular, the EGF ligands. However, broad-spectrum metalloprotease inhibitors tested for clinical use have failed as a result of indiscriminate blockade of substrate cleavage, leading to clinical side effects (11). Even recently developed selective ADAM inhibitors still affect the cleavage of many substrates (12). Modulation of the release of specific ADAM substrates has been impossible to date because it is unknown how cleavage specificity is regulated on the molecular level. It is therefore necessary to identify key signals that determine substrate specificity of cleavage.Ectodomain cleavage is made specific by a number of intracellular signals; e.g., by calcium influx, by activation of G protein-coup...

The discovery and subsequent isolation of tumor-initiating cells (TICs), a small population of highly tumorigenic and drug-resistant cancer cells also called cancer stem cells (CSCs), have revolutionized our understanding of cancer. TICs are isolated using various methodologies, including selection of surface marker expression, ALDH activity, suspension culture, and chemotherapy/drug resistance. These methods have several drawbacks, including their variability, lack of robustness and scalability, and low specificity. Alternative methods of purification take advantage of biophysical properties of TICs including their adhesion and stiffness. This review will provide a brief overview of TIC biology as well as review the most important methods of TIC isolation with a focus on biophysical methods of TIC purification.

Tumor-initiating cells (TICs), a subpopulation of cancerous cells with high tumorigenic potential and stem-cell-like properties, drive tumor progression and are resistant to conventional therapies. Identification and isolation of TICs are limited by their low frequency and lack of robust markers. Here, we characterize the heterogeneous adhesive properties of a panel of human and murine cancer cells and demonstrate differences in adhesion strength among cells, which exhibit TIC properties and those that do not. These differences in adhesion strength were exploited to rapidly (~10 min) and efficiently isolate cancerous cells with increased tumorigenic potential in a label-free manner by use of a microfluidic technology. Isolated murine and human cancer cells gave rise to larger tumors with increased growth rate and higher frequency in both immunocompetent and immunocompromised mice, respectively. This rapid and label-free TIC isolation technology has the potential to be a valuable tool for facilitating research into TIC biology and the development of more efficient diagnostics and cancer therapies.

In spite of major therapeutic advances, cancer relapse and low rates of patient response persist. This failure is due in part to a small subpopulation of tumor initiating cells (TICs) with stem cell like properties that are responsible for the growth of the tumor and the progression of metastasis. Currently, no efficient and reliable methods to isolate TICs for study exist. Our lab has developed a methodology to isolate cells based on their unique adhesion binding strength to a matrix. The novel technology (micro-Stem cell High- Efficiency Adhesion based Recovery [μSHEAR]) consists of a microfluidic device that applies varying degrees of detachment shear forces on cells. Using this device, human pluripotent stem cells and their progeny have been isolated with high reproducibility, yield (>97%), purity (95-99%), and survival (>95%) rates (Singh et al, Nature Methods 2013). The process is fast (10 min), label free, and scalable. The objective of this research is to isolate the rare TICs from the general cancer cell population by exploiting differences in adhesion strength. To study these differences, we measured the adhesion strength (adhesive signature) of a panel of breast cancer cell lines to fibronectin using a hydrodynamic assay. Based on this screen, we selected the MDA-MB- 231, MDA-MB-453, and MCF7 cell lines for purification of TICs using the uSHEAR technology. Briefly, microfluidic channels were sterilized and coated with fibronectin. MDA-MB- 231, MDA-MB-453 or MCF7 breast cancer cells were enzymatically disassociated, pipetted into the inlet reservoir, and cultured in the device for 24h before detachment experiments. Cells were exposed to different shear forces to selectively detach cell sub-populations. Recovered cell/colonies were counted, seeded into a mammosphere formation assay (MFA), and analyzed after 10 days The ability to form mammospheres is characteristic of TICs. After uSHEAR mediated separation of breast cancer cells into three fractions, the strongest adhering fraction consistently produced larger and more mammospheres. Furthermore, as the selection adhesive force for the adhered fraction was increased, greater increases in mammosphere number and size were observed. After 10 days, the mammospheres were disassociated and the number of cells quantified. A 5-15 fold increase in the final number of cells was observed in the strongly adherent fractions of cells, compared to 0.8-1.7 fold increase in the unsorted controls. Our results show that cancer cells with a higher mammosphere formation potential, a hallmark of characteristic TICs, have a higher adhesion strength. Furthermore, these cells can be enriched via adhesion based separation giving rise to more and larger mammospheres than their less adherent counterparts. Our results suggest that TICs could be separated based on adhesive forces. Future studies are aimed at characterizing the isolated cells as TICs and testing the isolation strategy in primary tumors. Citation Format: Efrain A. Cermeno, Austin P. Veith, Susan N. Thomas, Andres J. Garcia. Adhesive signature technology for tumor initiating cell purification in cancer research. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2494.