METHODS AND PRODUCTS TO TARGET, CAPTURE AND CHARACTERIZE STEM CELLS

CROSS REFERENCE TO RELATED APPLICATION

[01] This application claims the benefit of U.S. Provisional Application No.

61/018,157, filed 31 December 2007, entitled "METHODS AND PRODUCTS TO TARGET, CAPTURE AND CHARACTERIZE STEM CELLS", attorney docket no. LOU01 -G23-PRO, the contents of which are hereby incorporated by reference in their entirety, except where inconsistent with the present application.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[02] This invention was made with government support under R01 CA 122 383 awarded by the National institute of Health. The government has certain rights in the invention.

BACKGROUND

[03] Many methods for treating cancer are available. Those methods include surgery

(physical removal of the cancerous tissues), radiation therapy (killing cells by exposure to cell-lethal doses of radioactivity), chemotherapy (administering chemical toxins to the ceils), immunotherapy (using antibodies that target cancer cells and mark them for destruction by the innate immune system) and nucieic acid-based therapies (e.g., expression of genetic material to inhibit cancer growth). Such therapies take aim against ail tumor cells, but studies have shown that only a minor fraction of cancer cells have the ability to reconstitute and perpetuate the malignancy. If a therapy shrinks a tumor but misses these ceils, the cancer is likeiy to return [1],

[04] Moreover, in certain types of cancer it is now clear that only a tiny percentage of tumor cells have the power to produce new cancerous tissue, providing support for the theory that rogue stem-iike cells are at the root of many cancers. Because they are the

engines driving the growth of new cancer celis and are very probably the origin of the malignancy itself, these cells are called cancer stem cells. Additionally, cancer stem cells may be the oniy celis that can form metastases, the primary cause of death and suffering in patients. Targeting these cancer stem cells for destruction may be a far more effective way to eliminate the disease, as treatments that specifically target the cancer stem ceils couid destroy the engine driving the disease, leaving any remaining non-tυmorigenic ceils to eventually die off on their own [1].

[05] Stem ceils, however, cannot be identified based solely on their appearance, so developing a better understanding of the unique properties of cancer stem cells will first require improved techniques for isolating and studying these rare cells. Once their distinguishing characteristics are learned, the information can be used to target cancer stem ceils with tailored treatments. If scientists were to discover the mutation or environmental cue responsible for conferring the ability to self-renew on a particular type of cancer stem cell, for instance, that would be an obvious target for disabling those tumorigenic cells [1],

[06] Nucleolin [8] is an abundant non-ribosomal protein of the nucleolus, the site of nbosαrna! gene transcription and packaging of pre-ribosomal RNA. This 707 amino acid phosphoprotein has a multi-domain structure consisting of a histone-like N- terminus. a central domain containing four RNA recognition motifs and a glycine/arginine-hch C-terminus and has an apparent molecular weight of 110 kD. While nucleolin is found in every nucleated cell, the expression of nucieoSin on the ceil surface has been correlated with the presence and aggressiveness of neoplastic cells [33-

[07] Guanosine-rich oligonucleotides (GROs) designed for triple helix formation are known for binding to nucleolin [5]. This ability to bind nucleolin has been suggested to cause their unexpected ability to effect antiproliferation of cultured prostate carcinoma cells [6]. The antiproliferative effects are not consistent with a triplex-mediated or an antisense mechanism, and it is apparent that GROs inhibit proliferation by an alternative

mode of action, it has been surmised that GROs, which display the propensity to form higher order structures containing G-quartets, work by an aptamer mechanism that entails binding to nucleoϋπ due to a shape-specific recognition of the GRO structure. The binding to the ceil surface nucieolin then induces apoptosis.

[08] The correlation of the presence of cell surface nucieoiin with neopiastic celis has been made use of in methods for determining the neoplastic state of cells by detecting the presence of nucSeolin on the plasma membrane of the cells [3], This observation has also provided new cancer treatment strategies based on administering compounds that specifically targets nucieoiin [4],

SUMMARY

[09] in a first aspect, the present invention is a method for identifying cancer stem ceils, comprising reacting a piurality of cells comprising cancer stem cells with an anti- nucieolin agent to bind the anti-nucleolin agent to the cancer stem cells; and identifying the cancer stem cells that are bound to the anti-nucleoSin agent from remaining cells of the plurality of cells.

[10] in a second aspect, the present invention is a method for isolating cancer stem ceils, comprising reacting a piurality of cells comprising cancer stem cells with an anti- nucleolin agent to bind the anti-nucϊeoltn agent to the cancer stem cells; and separating the cancer stem cells that are bound to the anti-nucleolin agent from remaining cells of the plurality of cells,

[11] in a third aspect, the present invention is a method of profiling the genetic signature of a cancer stem cell, comprising isolating cancer stem cells; generating sequence reads of the genome of the cancer stem cells; aligning the sequence reads with a known genomic reference sequence; and analyzing variations between the sequence reads and the known genomic reference sequence.

[12] In a fourth aspect, the present invention is a method of identifying genes that are expressed in cancer stem cells, comprising generating a first gene expression profile of a sample of cancer ceils comprising the cancer stem celis; contacting the cancer ceils with an anti-nucleoiin agent to induce apoptosis in the cancer stem celis; generating a second gene expression profiie of the sample of cancer ceils; and identifying the genes having a reduced expression in the second gene expression profiie than in the first gene expression profiie.

[13] Sn a fifth aspect, the present invention is a method of treating leukemic bone marrow, comprising separating out cancer stem celis from the ieukemic bone marrow ex vivo, by reacting the Seυkemic bone marrow with an anti-nucieolin agent and removing the cancer stem cells bound to the anti-nucleolin agent,

[14] DEFINITIONS

[15] The phrase "cancer stem ceils" refers to cancer ceils capable of giving rise to multiple progeny.

[16] The phrase "differentiated cancer ceils" refers to cancer ceils that are not cancer stem ceils.

[17] The phrase "anti-nucleolin agent" refers to an agent that binds to nυcleoiin.

Examples include anti-nucleolin antibodies and certain guanosine-rich oiigonucieotides (GROs), Anti-nucleolin antibodies are weli known and described, and their manufacture is reported in Milier et al. [7]. Examples of anti-nucleoiin antibodies are shown in Tabie 1. GROs and other oligonucleotides that recognize and bind nucleoSin can be used much the same way as are antibodies. Examples of suitable oligonucleotides and assays are also gsven in Miiler et al. [7]. In some cases _r incorporating the GRO nucleotides into larger nucleic acid sequences may be advantageous; for example, to facilitate binding of a GRO nucleic acid to a substrate without denaturing the nucleoϊin-

binding site. Examples of oligonucleotides are shown in Table 2; preferred oligonucleotides include SEQ IDs NOs; 1-7; 9-16; 19-30 and 31 from Table 2.

[20]

BRIEF DESCRIPTION OF THE DRAWINGS

[21] Figure 1 illustrates the results of an in vivo xenograft experiment in nude mice, in which cancer ceils (A549 celis} _: pre~treated with a nucieoltrvbinding aptamer (AGRO 100), have decreased tυmorigenicity in the immunocompromised mice, as compared to cancer cells which were not treated,

[22] Figure 2 illustrates the results of an in vivo xenograft experiment in nude mice, in which cancer cells (HCT116 cells), pre-treated with a nυcleolin-binding aptamer (AGRO 100), have decreased tumorigenicity in the immunocompromised mice, as compared to cancer cells which were not treated.

[23] Figures 3 and 4 illustrate the results of aldefluor staining of DU 145 ce!ls _: untreated or treated, respectively, with a nucleoSin-binding aptamer. High expression of aldehyde dehydrogenase (ALDH), which reacts with the aldefluor to produce a bright fluorescence, is associated with cancer stem cells. The fluorescence of the untreated cells (63.9% ALDH+ versus the control sample), as compared to the fluorescence of the

treated cells (27.9% ALDH+ versus the control sampie), indicates that the treated cells contain fewer cancer stem cells.

[24] Figure 5 illustrates the results of aldefiuor staining of HCT116 ceils treated with a nucieσiin-binding apfarner. High expression of aldehyde dehydrogenase (ALDH), which reacts with the aldefiuor to produce a bright fluorescence, is associated with cancer stem ceils. The fluorescence of untreated cells (70.4% ALDH+ versus the contro! sample, data not shown), as compared to the fluorescence of treated celis (61.7% ALDH+ versus the control sample), indicates that the treated cells contain fewer cancer stem ceils.

[25] Figures 6 and 7 iilustrate the effect of treatment with a nucleolin-binding aptamer, on cancer-stem-celi enriched subpopuiations of A549 ceils. These cancer-stem -ceil enriched subpopuiations are identified by the fact that they expei a fluorescent dye, with the least fluorescent subpopuiation ("'bottom of SP") presumed to be the most stem cell- like. Figure 6 shows the results of a control experiment using buffer, resulting in a subpopuiation SP = 28.08%, and the most fluorescent portion of the subpopuiation ("top of SP") being 11.09%, and the bottom of SP - 4.97%; Figure 7 shows the results of treatment with a nucleolin-binding aptarner, resulting in a subpopuiation SP = 21.75%, with the top of SP = 12.83%, and the bottom of SP = 1.20%.

DETAILED DESCRIPTION

[26] The present invention makes use of the discovery that cancer stem ceils are characterized by high ieveis of nucieolin (in particular ceil surface or cytoplasmic nucieoiin) as compared to differentiated cancer celis. Therefore, the binding of an anti- nucieoiin agent to a cancer cell is indicative that the eel! is cancer stem cell.

[27] During clinical trials that employ nucleolin-binding GROs in the treatment of prostate cancer, it was discovered that the clinica! response to the GROs is very unusual. A single dose of GROs may have no initial effect, but over several months

may cause complete tumor regression without any further treatment. Without being bound to any particular theory, this response is what would be expected from a therapy targeting cancer stem cells. These observations were buttressed by gene expression studies on cultured prostate carcinoma cells; following treatment with GROs. the expression of genes known to be active in stern ceils was specifically down-regulated, while the expression of genes active in quiescent cells was not.

[28] The binding of an anti-nucleαiin agent allows one to specifically differentiate between cancer stem cells and differentiated cancer cells. Various techniques can therefore be used to identify and isolate cancer stem cells by taking advantage of the fact that the cancer stem cells will bind to the anti-nucleoSin agent. Also, since treatment with a GRO specifically targets cancer stem cells for apoptosis, the genetic signature of cancer stem ceils can be profiled and genes that are expressed in cancer stem cells can be identified, by comparing a sample of cancer cells before and after treatment with an anti-nucleolin agent.

[29] The present invention provides methods for identifying cancer stem cells by binding of an anti-nucleoiin agent. Samples of cancer ceils, optionally isolated from a subject, are reacted wrth an anti-nucieohn agent. Procedures for detecting and/or identifying the cancer stem cells in a sample can use an anti-nucleoSin agent; these agents may be directly labeled or, when bound to a cell, detected indirectly.

[30] Cells bound to anti-nucleolin agents may be detected by known techniques. For example, immunofluorescence employs fluorescent labels, while other cytologica! techniques, such as histochemical. immunohistochernical and other microscopic (electron microscopy (EM), immunoEM) techniques use various other labels, either coiorimetric or radioactive The techniques may be carried out using, for example, anti- nucleolin agents conjugated with dyes, radio isotopes, or particles. Alternatively, an antibody specific for the anti-nucSeolin agent may be used to label the cell to which the anti-nucleolin agent is bound.

[31] Also provided are methods for isolating cancer stem cells. Samples of cancer cells are reacted with an anti-πυcleoliπ agent to bind the aπti-πucleolin agent selectively to the cancer stem cells. The cancer stem cells that are bound to the aπti-nucleolin agent are then separated from the remaining celis. Cells bound to the anti-nucleolin agent may be separated by techniques that are well known. For example, in immmunopanning-based methods, an anti-nucSeolin agent is bound to a substrate, for instance the surface of a dish, filter or bead; cells binding to the anti-nucleolin agent adhere to the surface, while non-adherent cells can be washed off. Alternatively, the surface may be fυnctionalized with an agent that binds an anti-nudeolin agent; the cells of the sample are reacted with the anti-nucleolin agent, and then subsequently the cells are reacted with the surface. The celis that bind to the anti-nucleolin agent will therefore also adhere to the surface. This may be accomplished, for example, by using an anti- nucieolin agent-biotin conjugate, and functionalizing the surface with streptavidin,

[32] In methods based on fluorescence-activated cell-sorting, a sample of cancer cells is worked into a suspension and reacted with a fiuorescent-tagged anti-nucleolin binding agent. The cell suspension is entrained in the center of a stream of liquid. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell being in a droplet. Just before the stream breaks into droplets the flow passes through a fluorescence measuring station where the fluorescence of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately prior fluorescence intensity measurement and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge, thereby isolating the cells that are bound to the anti-nucleoSin agent.

[33] The invention also provides methods for profiling the genetic signature of cancer stem cells. Cancer stem cells are isolated as illustrated above, and sequence reads of

the genome of the cells are generated. The sequence reads are aligned with known genomic reference sequences and variations between the sequence reads and the references sequences are analyzed,

[34] Furthermore, methods for identifying genes that are expressed in cancer stem cells are also provided, A first gene expression profile of a sample of cancer cells is generated by a well known method, such as by using a RT-PCR array. The sample is then treated with an anti-nucSeolin agent to bind the cancer stem cells, and induce apoptosis, for example using AS 1411 (also known as AGRO 100, or GRO26B in Table 2). Following this treatment, a second gene expression profile of the sample is generated. The first and second profiles are then compared, and genes which have a reduced expression in the second profile, as compared to the first profile, are identified as those of the cancer stem cells. The following tables (Tables (A), (B) ₁ (C) and (D)), describe the results of such an experiment carried out with prostate cancer cells, using AS1411 as the anti-nucleolin agent and using a RT-PCR array for generating the gem expression profiles

[39] Two in vivo xenograft experiments were carried out in nude mice, in which cancer cells (A549 cells or HCT116 cells) were either pre-treated with a nucleolin- binding aptamer (AGRO 100) or left untreated. In a T150 flask, the cancer cells in DMEM (+ 10% heat-inactivated FBS + 1 % penicillin/streptomycin) were grown to 100% confluence. The cells were split 1 :10, to make two new T150 flasks of cancer cells. These cells were grown to 50-70% confluence. Later, the media was removed, and 2OmL of fresh media was added to each flask. To the experimental fiask {+), 0,4mL of 50OuM AS 1411 from frozen stock was added (1OuM final concentration). To the control flask (-), 0,4ml of 1OmM potassium phosphate was added (1OmM potassium phosphate was used to prepare the AS1411 frozen stock). The flasks were incubated for 18 hours at 37 ⁰ C, 5% CO, Later, the media was removed, and the cells were washed twice with PBS. The cells were then trypsinized, harvested with 1OmL of media, and counted.

Next the ceils were centrifugecS, the supernatant removed, and the cells resuspended in PBS to make a final concentration of 10 ⁷ celis per mL (~ 10 ⁶ ceils / 10OuL).

[40] The ceils were injected (10OuL subcutaneous injections) into each group of five femaie nude mice, with 10 ⁶ (-} ceISs injected into the ieft flank, and 10 ⁶ (+) celis injected into the right fiaπk. Tumor growth was then monitored. Figure 1 iilustrates the results of the in vivo xenograft experiment, using A549 ceils: the cells pre-treated with a nucleolin- binding aptamer (AGRO 100) have decreased tumorigenicity in the immunocompromised msce, as compared to the cancer celis which were not treated. Figure 2 illustrates the results of the in vivo xenograft experiment using HCT116 celis. again, the celis pre-treated with a nucieoiin-binding aptamer (AGRO 100) have decreased tumorigenicity in the immunocompromised mice, as compared to the cancer ceils which were not treated.

[41] Two aldefiuor staining experiments were carried out, in which cancer celis

(DU 145 cells or HCT116 cells) were either treated with a nucieoiin-binding aptamer (AGRO 100} or left untreated. High expression of aldehyde dehydrogenase (ALDH) is associated with cancer stem ceils. Aldefiuor staining may be used to identify celis with high expression of ALDH, because the enzyme reacts with the aldefiuor to produce a bright fiuorescence.

[42] in two T150 fiasks, DU145 prostate cancer celis in DMEM (+ 10% heat- inactivated FBS + 1% peniciiSin/streptomycin) were grown to -80% confluence. Similarly, in two T150 flasks. HCT116 colon cancer ceils in McCoy's (+ 10% heat- inactivated FBS + 1 % penicillin/streptomycin) were grown to -80% confluence. Later, the media was removed, and 15 mL of fresh media was added to each flask. To the experimental flasks (+), 0.3mL of 50OuM AS1411 from frozen stock was added (1OuM final concentration). To the control flasks {-}, 0,3mL of 1OmM potassium phosphate was added (1OmM potassium phosphate was used to prepare the AS1411 frozen stock). The flasks were incubated for 18 hours at 37 ⁰ C, 5% CO. The Aldefiuor Assay Buffer

and DEAB inhibitor were removed from refrigerator, and allowed to warm to room temperature. An aliquot of aidefluor at -2O ⁰ C was thawed on ice.

[43] Two 12 x 75mm flow cytometry lubes were labeled, one as control and the other as test. The media was removed from the flasks, and the cells were washed twice with PBS. Next, 3mL of TrypLE Express (GiBCO) was added to each flask. The cells were incubated for about 5 min at 37 ⁰ C until the cells were completely freed from the flasks. 5 ml of media was added to neutralize the TrypLE Express, and the cells were pipetted up and down to break clumps, and then counted,

[44] In the tube labeled "test," 2.5 x 10 ⁶ cells were placed. The tube was centrifuged

(Sorvall RT7 Plus) for 5 min at 1000 rpm _r at room temperature, and the supernatant was removed from the cell pellet. 2.5ml of Assay Buffer was added to make a final cell concentration of 10 ⁶ cells/mL To the tube labeled "control," 7.5uL DEAB was added. To the tube labeled "test" 12,5uL of aidefluor reagent (5uL per ml) was added. Without delay, the contents were mixed with a vortex at half speed, and then 0.5mL of this sample was placed in tube labeled "control". Another 0,5mL was removed from the "test" tube and place in the "Pi" tube. Ail tubes were sealed with parafilm, and incubated in a 37 ⁰ C water bath for 30 minutes, with occasional mixing.

[45] The tubes were again centrifuged, except at 4 ⁰ C rather than at room temperature. The supernatant was aspirated from the cell pellet. The cells were resuspended in cold Assay Buffer to make a final concentration of 10° ceSls/mL (0.5m L to "control" and "Pi," and 1.5mL to "test"). The cells were kept on ice until they were analyzed.

[46] Figures 3 and 4 illustrate the results of the aidefluor staining of DU145 ceils, untreated or treated _r respectively, with a nucleolin-binding aptamer. The fluorescence of the untreated cells as compared to the control sample with DEAB inhibitor showed an ALDH+ population of 63.9%, while the fluorescence of the treated cells as compared to the control sample showed an ALDH+ population of 27.9%. Pretreatment with a

nucleoiin-binding aptamer decreased the ALDH+ population in the DU 145 cells by 56% (from 63.9% to 27.9%), indicating that the treated cells contain fewer cancer stem cells.

[47] Figure 5 illustrates the results of aldefiuor staining of HCT116 ceils treated with a nυcieoiiπ-binding aptamer. The fluorescence of the untreated cells as compared to the control sample with DEAB inhibitor, showed an ALDH+ population of 70.4% (data not shown), while the fluorescence of the treated cells as compared to the contro! sample showed an ALDH+ population of 61.7%. Pretreatment with a nucleolin-biπding aptamer decreased the ALDH+ population in the HCT116 cells by 12% (from 70.4% to 61.7%), indicating that the treated cells contain fewer cancer stem cells,

[4S] An experiment was carried out to determine the effect of treatment with a nucSeolin-binding aptamer on cancer-stem-ceil enriched subpopulations of A549 cells. These cancer-stem -eel I enriched subpopulations are identified by the fact that they expel a fluorescent dye by virtue of ABC-type drug efflux pumps and therefore are in a dye-negative "side population" (SP); the least fluorescent subpopuiation ("bottom of SP") is presumed to be the most stem celS-Sike.

[49] in two T150 flasks. A549 lung cancer ceils in DMEM ( ^■ * ^■ 10% heat-inactivated

FBS + 1 % peniciilsn/streptomycin) were grown to ~80% confluence. Later, the media was removed, and 15 mL of fresh media was added to each flask. To the experimental flasks {+), 0.3mL of 50OuM AS1411 from frozen stock was added (1OuM final concentration). To the contro! flasks (-), 0.3mL of 1 OmM potassium phosphate was added (1OmM potassium phosphate was used to prepare the AS1411 frozen stock). The flasks were incubated for 18 hours at 37 ⁰ C, 5% CO. The media was removed from the flasks, and the cells were washed twice with PBS. Next, 3mL of TrypLE Express was added to each flask to harvest the cells, and then 7m L of media added and the ceils counted. The cells were centrifuged to remove supernatant, and resuspended in pre-warmed DMEM (+ 10% heat-inactivated FBS + 1 % penicillin/streptomycin) to make a final concentration of 10 ⁶ celis/mL Up to SmL of the cell suspension (no more than 5 million cells per tube) was placed in 15 mL Falcon tubes wrapped in foil. Then, 50 uL of

verapamil was added to the control samples (1 OuL per ml). With the Sights off, 25 uL of Hoechst dye was added to the stained samples (5uL per ml). The tubes were incubated for 90 minutes in a 37 ⁰ C water bath _r while mixing the tubes reguiarly by inverting.

[50] From this point on, the cells were kept cold and protected from Sight. The lubes were again centrifuged, except at 4 ⁰ C rather than at room temperature. The supernatant was aspirated from the ceil pellet. The ceils were resuspeπded in 500 uL of coid HBSS ⁺ (from a 4 ⁰ C refrigerator), 2uL of PS was added to each sample, and the ceils were kept on ice untii they were analyzed.

[51] The resuits from this experiment are shown in Figures 6 and 7. Figure 6 shows the resuits of the control experiment using buffer, resulting in a subpopulation SP = 28.08%, with the most fluorescent portion of the subpopulation ("top of SP") being 11.09%, and the bottom of SP = 4.97%, Figure 7 shows the resuits of treatment with a nucleolin-binding aptamer, resulting in a subpopulation SP ~ 21.75%. with the top of SP - 12.83%, and the bottom of SP = 1.20%.

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