Method of enriching rare cells

#The present invention relates to a method of enriching rare cells in a fluid containing a mixture of rare cells and non-rare cells, and particularly to a method of enriching rare non-blood cells such as cancer cells, from bodily fluids, such as blood.

#There has been a growing interest in enriching rare cells (e.g., for subsequent isolation and characterization) over the past several years. This may be attributed at least in part to the recognition that rare cells, such as cancer cells, can provide information that is helpful in the diagnosis and/or treatment of various medical conditions.

#The desire to enrich cancer cells is based in part on the knowledge that a majority of cancer deaths occur due to the metastasis of tumors. As such, the presence of carcinoma cells in the peripheral blood is an indication of cancer cell spread, and enriching such cancer cells would be of great diagnostic benefit. This need is particularly acute in prostate cancer, wherein approximately two-thirds of such cancers are clinically localized at the time of diagnosis, but only about half of these prove to be confined to the prostate at the time of surgery. Thus, nearly one-third to one-half of cancers have spread beyond the prostate when first identified, cancers which could be detected at any earlier stage if accurate, highly sensitive enrichment methods were available.

#FIG. 1 schematically depicts a density gradient column before (on the left) and after (on the right) centrifuging a fluid sample (e.g., blood). Four regions are formed after centrifugation: Plasma I, Interface I, Gradient I, and Cell Pellet I. Interface I and Gradient I are combined to provide a first fluid including an increased concentration of rare cells, referred to below as the Collection I fluid. Plasma I and Pellet I can be combined and centrifuged to form another column having four regions.

#FIGS. 2A and 2B schematically depict centrifuging the combined Plasma I and Pellet I (from FIG. 1) on a single density gradient column (FIG. 2A) or a double density gradient column (FIG. 2B). The schematics illustrate the columns before (on the left) and after (on the right) centrifugation. Four regions are formed after centrifugation: Plasma II, Interface II, Gradient II, and Pellet II. Gradient II and Interface II will be combined to provide a second fluid including an increased concentration of rare cells, referred to below as the Collection II fluid.

#FIG. 3 schematically depict another embodiment of the invention, wherein a double density gradient column can be utilized to form six regions after centrifugation. The left side of FIG. 3 illustrates the double density gradient column and fluid sample (e.g., blood) before centrifugation, and the right side illustrates the column after centrifugation. Six regions are formed after centrifugation: Plasma, Interface I, Gradient I, Interface II, Gradient II, and Pellet. Interface I and Gradient I will be combined to form the Collection I fluid, and Interface II and Gradient II will be combined to form the Collection II fluid. The Collection I and II fluids each have an increased concentration of rare cells.

#The present invention provides a sensitive, economical, and reproducible method for enriching rare cells in a fluid comprising rare and non-rare cells. In accordance with the invention, a fluid comprising rare and non-rare cells is subjected to density gradient separation, before producing at least one fluid comprising an increased concentration of rare cells. The fluid comprising an increased concentration of rare cells is subjected to a "negative selection process" comprising contacting the fluid with an agent which binds the non-rare cells. The bound non-rare cells are then separated from the fluid, providing a fluid enriched with the rare cells. The rare cells can be further processed, e.g., to identify, characterize, and/or culture the cells. For example, the rare cells can be identified and characterized to detect one or more types of cancer. Embodiments of the present invention provide for monitoring the progress, or regression, of cancer during or after therapy, and are particularly useful for monitoring prostate cancer in men.

#In an embodiment, a fluid comprising rare and non-rare cells is subjected to density gradient separation, before producing a first fluid comprising an increased concentration of rare cells and a second fluid comprising an increased concentration of rare cells. This first fluid and/or second fluid is subjected to the negative selection process comprising contacting the fluid(s) with an agent which binds the non-rare cells. The bound non-rare cells are then separated from the fluid(s), providing the fluid(s) enriched with the rare cells.

#Additionally, since embodiments of the method according to the invention can be carried out while minimizing stress to those rare cells that are fragile and/or sticky, the rare cells can be recovered essentially unscathed. This is especially desirable, as the live recovered rare cells have a variety of uses, e.g., for studies of the whole cell and/or cell culturing. Moreover, embodiments of the method allow different forms of rare cells in the same sample (e.g., "light" and "heavy" rare cells) to be processed differently, thus allowing a great proportion, if not substantially all, of the rare cells to be recovered, while reducing the presence of the non-rare cells in the rare cell-enriched fluid.