Quantitative Analysis of a Single Cell, Intracellular Paramagnetic Metal Concentration in Cancer Cell Lines

Iron is an indispensable requirement for the activity of many vital biochemical processes such as cellular energetics, signaling, reactive oxygen species homeostasis, and DNA synthesis. The increased energy demand of the uncontrolled cancer cell division combined with the less efficient cellular energy conversion mechanism results in the increased demand for iron by cancer cells. The increased concentration of superoxide dismutase (that requires manganese for its function) in certain cancer cells has also been reported. Quantitative analysis of these two paramagnetic metals (iron or manganese) in a single cell would enable investigations of the relationship between a pathophysiological increase in the intracellular paramagnetic metal compounds and cancer cell metabolism.

In this study, the single cell iron and/or manganese concentration in eight cancer cell lines ((U937, K562, HL60, Hep 3B, Hep G2, CAL 27, Detroit 562, HeLa, all from ATCC, Manassas, VA) was measured by cell magnetophoretic mobility (MM) analysis using cell tracking velocimetry (CTV), in which a permanent magnet assembly producing a nearly constant magnetic energy density gradient, Sm (140 TA/mm2) was employed. MM is directly proportional to the cell magnetic susceptibility, which is a function of the intracellular high-spin iron or manganese species. We have shown that CTV is capable of measuring MM of the red blood cells as related to the heme spin state of the hemoglobin, Hb (high spin for deoxygenated Hb, null spin for oxygenated Hb). For comparison, the bulk iron and manganese concentration in each cell suspension was determined by inductively coupled plasma mass spectrometry (ICP-MS). The effect of a soluble iron compound addition (500 µg/mL Fe(NO3)3) to cell culture media on the intracellular iron concentration was also studied. The intracellular iron storage protein, ferritin, was imaged by transmission electron microscopy (TEM) by relying on its electron-dense crystalline ferrihydrite core.

The results of MM distribution analysis revealed the existence of small subpopulations (1-2%) in K562 and HeLa cell lines with high iron concentration and high intrinsic magnetic susceptibility, further confirmed by their isolation (without labeling) using a commercial magnetic separator (MiniMACS magnet and MS column, Miltenyi Biotec, Auburn, CA). Extra iron compound addition to cell culture media significantly increased MM of HeLa cell without altering their growth rate, viability, and cell size. TEM result shows that this MM increase was caused by the iron metabolism and storage in ferritin rather than iron cell surface adhesion. The effect was dependent on the cell types. Such cell metabolism-related soluble iron transfer into intracellular compartment establishes the possibility of an alternative magnetic cell labeling to immunomagnetic labeling, and the possibility of a label-free magnetic cell separation based on their natural, intrinsic magnetization.