Rapid Recognition and Isolation of Live Colon Cancer Stem Cells by Using Metabolic Labeling of Azido Sugar and Magnetic Beads

Abstract

New approach for colon cancer stem cells (CSCs) recognition and isolation is reported. Colon CSCs are responsible for colonic tumor growth, metastasis, and resistance for radio-/chemotherapies. An accurate identification and isolation method is critical for understanding and characterization of these cells. In our work, we recognized CSCs' population from colon cancer cells by using metabolic labeling of azido sugar based on the quiescent nature of these cells, which differed fundamentally from previously described methods by using specific cellular markers to recognize and isolate CSCs. Later the putative CSCs were isolated by using commercially available magnetic beads. The isolated cells population had much higher sphere formation efficiency, soft-agar colony formation efficiency, and an mRNA level of colon stem cells marker Lgr5 than the leftover population. Our method provides a new avenue and a general strategy for recognition and isolation of CSCs, which shows great potential for further use in both the fundamental research of CSCs and clinical tests.

Safety and Optimization of Metabolic Labeling of Endothelial Progenitor Cells for Tracking

“…In addition, the reaction produces very few toxic and non-toxic byproducts and therefore, metabolic labeling has advantages that apply to in vivo studies. Till now, the metabolic labeling technique in stem cells has been mostly used for detecting glycoprotein markers of mesenchymal stem cell differentiation 31 , for identifying or isolating live colon cancer stem cells 32 and for quantifying protein abundance in embryonic stem cells (ESCs) 33 . However, the cellular mechanisms by which modified glycosylation due to metabolic agents are not completely understood 34 .…”

Optomicrofluidic detection of cancer cells in peripheral blood via metabolic glycoengineering

“…The rate of cell metabolism has a significant influence on azide sugar expression. 27 Cancer cells have an enhanced requirement of energy to accomplish their accelerated biological functions such as fast proliferation, vigorous migration, and proactive invasion. 37 High metabolism is an essential aspect of tumorigenesis and the progression of the disease, which differentiates cancer cells from normal cells.…”

“…Instead of relying on naturally occurring surface molecules, artificial surface molecules can be introduced into the cells via the surface glycoengineering technique, which depends on the cell metabolism rate. 27 Glycoengineering techniques have been used for introducing azide molecules on the surface of different types of cancer cells, mainly for imaging or drug delivery applications. [26][27][28] Triple-negative breast cancer is one of the most challenging tumors to target as it lacks all three receptors, estrogen receptor, progesterone receptor, and HER2, generally found in breast cancer cell lines.…”

“…27 Glycoengineering techniques have been used for introducing azide molecules on the surface of different types of cancer cells, mainly for imaging or drug delivery applications. [26][27][28] Triple-negative breast cancer is one of the most challenging tumors to target as it lacks all three receptors, estrogen receptor, progesterone receptor, and HER2, generally found in breast cancer cell lines. It has been shown that even triplenegative breast cancer tumors in live mice could be targeted through glycoengineering, which highlights the applicability of the technique.…”

“…N-Azidoacetyl-mannosamine-tetraacylated (Ac 4 -ManNAz) undergoes metabolism forming N-azidoacetyl neuraminic acid, which is integrated into glycan, expressing surface-azide groups that give rise to fluorescence signal emission when treated with fluorescently labeled dibenzocyclooctyne (DBCO). Cancer cells are incubated in Ac 4 ManNAz, instead of being cultured with it for three days as done in previous studies, 27,29 making the protocol amenable to in situ tagging of CTCs. The effects of concentrations of the compounds used for metabolic labeling and incubation time on the fluorescence signal intensity are studied which showed that the proposed tagging protocol is comparable to conventional CTC tagging protocols.…”