ATP-based cell sorting and hyperproliferating cancer stem cells

Abstract

High mitochondrial ATP is a metabolic feature that gives cancer cells excessive proliferation, dryness, non-anchorage dependence, antioxidant capacity and multidrug resistance. Under this method, intracellular ATP levels can be used as metabolic biomarkers for identifying, separating and purifying invasive and overproliferation cancer stem cells ("CSC") phenotypes. In addition, ATP can be combined with other CSC markers (such as CD44 or ALDH activity) to beneficially classify CSC groups into subgroups. For example, the non-anchorage-dependent growth level shown by high ATP / high CD44CSC subgroups is twice that of low ATP / high CD44CSC subgroups. It is also disclosed that mitochondrial ATP synthesis involves the dryness, transfer and detection of circulating tumor cells ("CTC"), and five ATP-related transfer gene features (ABCA2, ATP5F1C, COX20, NDUFA2 and UQCRB). The gene features of this method can be used to identify CSCs with significantly increased cell migration and invasion ability in vitro and spontaneous metastasis in vivo. This approach also provides a cellular platform for systemically targeting the stemness, multidrug resistance, and metastasis of cancer cells.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 900,139, filed September 13, 2019, and is incorporated herein by reference in its entirety.

[0003] field

[0004] The present disclosure relates to ATP-based cell sorting to identify, isolate and treat metabolically hyperactive, invasive and hyperproliferative cancer stem cell ("CSC") phenotypes and to prevent or reduce the likelihood of metastasis.

[0005] background

[0006] Researchers are continually working to develop new anticancer therapies. Traditional cancer therapies (e.g., radiation, alkylating agents such as cyclophosphamide, and antimetabolites such as 5-fluorouracil) attempt to selectively detect and eradicate rapidly growing cancer cells by interfering with cellular mechanisms involved in cell growth and DNA replication. Other cancer therapies use immunotherapies (e.g., monoclonal antibodies) that selectively bind to mutated tumor antigens on rapidly growing cancer cells. Unfortunately, tumors often recur at the same or a different site after these treatments, suggesting that not all cancer cells have been eradicated. Relapse may be due to inadequate chemotherapy dosing and / or the emergence of cancer clones that are resistant to treatment. Therefore, new cancer treatment strategies are needed.

[0007] Advances in mutation analysis have allowed for in-depth studies of genetic mutations that occur during cancer development. Despite an understanding of the genomic landscape, modern oncology still struggles to identify the major driver mutations of different cancer subtypes. The harsh reality seems to be that each patient's tumor is unique and a single tumor may contain multiple different clonal cells. Therefore, a new approach that emphasizes the commonalities between different cancer types is needed. Targeting metabolic differences between tumor cells and normal cells is expected to become a new cancer treatment strategy. Analysis of transcriptional profiling data from human breast cancer samples revealed that more than 95 elevated mRNA transcripts were associated with mitochondrial biogenesis and / or mitochondrial translation. In addition, more than 35 of the 95 upregulated mRNAs encode mitochondrial ribosomal proteins (MRPs). Proteomic analysis of human breast cancer stem cells also revealed a significant overexpression of several mitochondrial proteins and other proteins associated with mitochondrial biogenesis.

[0008] Mitochondria are extremely dynamic organelles that continuously divide, elongate, and interconnect to form tubular networks or fragmented granules to meet cellular needs and adapt to the cellular microenvironment. The balance of mitochondrial fusion and fission determines the morphology, abundance, function, and spatial distribution of mitochondria, thereby affecting a large number of important biological processes that depend on mitochondria, such as ATP production, mitophagy, apoptosis, and calcium homeostasis. In turn, mitochondrial dynamics can be regulated by mitochondrial metabolism, respiration, and oxidative stress. Therefore, it is not surprising that an imbalance in fission and fusion activities has adverse effects on several pathological conditions, including cancer. Although cancer cells often display fragmented mitochondria, and enhanced fission or reduced fusion are often associated with cancer, a comprehensive understanding of how mitochondrial dynamics affects tumorigenesis is still needed.

[0009] Intact and enhanced metabolic function is necessary to support the elevated bioenergetic and biosynthetic demands of cancer cells, particularly as they progress toward tumor growth and metastatic spread. Not surprisingly, mitochondrial-dependent metabolic pathways provide an important biochemical platform for cancer cells by extracting energy from several fuel sources.

[0010] Cancer stem-like cells are relatively small subpopulations of tumor cells that share common features with normal adult stem cells and embryonic stem cells. Therefore, CSCs are considered to be the "main biological cause" of tumor regeneration and systemic spread of life, leading to clinical features of tumor recurrence and distant metastasis, and ultimately leading to treatment failure and premature death in cancer patients receiving chemotherapy and radiotherapy. There is evidence that CSCs also play a role in tumor initiation, as isolated CSCs have been experimentally characterized as tumor-initiating cells (TICs) in preclinical animal models. Since approximately 90% of all cancer patients worldwide die prematurely from metastatic disease, the need for the development of new therapeutic approaches that effectively target and eradicate CSCs is urgent and clinically unmet. Most conventional therapies do not target CSCs and typically increase the frequency of CSCs in primary tumors and distant sites.

[0011] Recently, energy metabolism and mitochondrial function have been implicated in certain dynamics involved in the maintenance and proliferation of CSCs, a unique subpopulation of cells within the tumor mass that are involved in tumor initiation, metastatic spread, and resistance to anticancer therapy. For example, CSCs exhibit an unusual and unique increase in mitochondrial mass, as well as enhanced mitochondrial biogenesis and higher activation of mitochondrial protein translation. These behaviors suggest a strict dependence on mitochondrial function. Consistent with these observations, elevated mitochondrial metabolic function and OXPHOS have been detected in CSCs in multiple tumor types.

[0012] An emerging strategy to eliminate CSCs takes advantage of cellular metabolism. CSCs are one of the most dynamic cancer cells. Under this method, metabolic inhibitors are used to induce ATP depletion and starve CSCs. So far, the inventors have identified many FDA-approved drugs with off-target mitochondrial side effects that have anti-CSC properties and induce ATP depletion, including, for example, the antibiotic doxycycline, which acts as an inhibitor of mitochondrial protein translation. Doxycycline is a long-acting tetracycline analog that is currently used in particular to treat various forms of infection, such as acne, rosacea, and malaria prevention. In a recent Phase II clinical study, preoperative oral doxycycline (200 mg per day for 14 days) reduced the CSC burden of patients with early breast cancer by 17.65% to 66.67%, with a positive response rate of nearly 90%.

[0013] However, certain shortcomings limit the use of single anti-mitochondrial agents in cancer therapy, as adaptive mechanisms can be employed in tumor masses to overcome the lack of mitochondrial function. These adaptive mechanisms include, for example, the ability of CSCs to switch from oxidative metabolism to alternative energy pathways in a multidirectional process of metabolic plasticity driven by intrinsic and extrinsic factors within the tumor cell and in the surrounding niche. Notably, manipulation of this metabolic flexibility in CSCs may become therapeutically advantageous. What is needed next is therapies that prevent these metabolic shifts or otherwise exploit them to inhibit cancer cell proliferation.

[0014] Adenosine 5'-triphosphate (ATP) is the bioenergetic "currency" of all living cells and organisms. Chemically, ATP is a nucleoside triphosphate, which contains adenine, ribose, and three phosphate groups. ATP is cleaved at its terminal phosphate group to produce two major reaction products, ADP and inorganic phosphate (Pi), thereby releasing high levels of stored energy. In eukaryotic cells, mitochondria generate large amounts of ATP through the TCA cycle and oxidative phosphorylation (OXPHOS), while glycolysis produces smaller amounts of ATP. Mitochondrial dysfunction induces ATP depletion, leading to mitochondrial-driven apoptosis (cell death).

[0015] In MCF7 breast cancer cells, mitochondria-driven OXPHOS contributes 80% of ATP generation, while glycolysis contributes the remaining 20%. Thus, cancer cells, like normal cells, remain highly dependent on mitochondria for ATP production. However, how ATP levels in cancer cells contribute to "stemness" and cell cycle progression, as well as their ability to undergo anchorage-independent growth, a hallmark of metastatic spread, remains largely unknown.

[0016] Due to the central importance of ATP as a barometer of cellular metabolism, many luminescent and fluorescent probes have been developed to measure and track ATP levels in response to various cellular stimuli. For example, ATP-Red 1 (CAS#: 1847485-97-5, IUPAC name: [2-[3',6'-bis(diethylamino)-3-oxospiro[isoindol-1,9'-xanthene]-2-yl]phenyl]boronic acid) is a vital dye that fluoresces only when bound to ATP and does not recognize ADP or other nutrients. ATP-Red 1 allows for dynamic visualization of ATP levels in living cells and tissues.

[0017] The purpose of this disclosure is to describe a feasible ATP depletion strategy to target and eradicate even the "fittest" cancer cells.

[0018] It is another object of the present disclosure to describe unique compositions of cells having a specific hyperproliferative phenotype.

[0019] Another object of the present disclosure is to identify novel anti-cancer therapeutic approaches involving novel drug compounds that metabolically starve CSCs by targeting mitochondria and driving ATP depletion.

[0020] Overview

[0021] The present method describes the use of fluorescent ATP imaging probes to metabolically grade cancer cell populations and isolate overproliferative cell subpopulations. The resulting composition can be used for a variety of advantageous purposes, ranging from rapid drug development and screening to predicting and preventing metastasis and drug resistance. The present method also provides a 5-gene signature prognosis of cancer metastasis, as well as methods for metabolic classification of cancer cells and diagnosis and prevention of metastasis.

[0022] Bioenergetic cell "stratification" using ATP-based biomarkers can be used to isolate "fittest" cancer cells for identification, diagnosis, treatment, and therapeutic drug development. In particular, fluorescent ATP imaging probes such as Biotracker ATP-Red 1 can be used to stain cell populations, and if desired, the resulting ATP-based fluorescence can be used to metabolically stratify the cell population into high ATP subpopulations, bulk subpopulations, and low ATP subpopulations. Using this new approach, the data disclosed herein include the first evidence that high levels of mitochondrial ATP are a major determinant of aggressive cancer cell behavior, including spontaneous metastasis.

[0023] There is a great deal of phenotypic diversity and metabolic heterogeneity within cancer cell populations. This heterogeneity allows the “fittest” cancer cells to evade current therapeutic modalities, leading to tumor recurrence and distant metastasis secondary to drug resistance.

[0024] High intracellular ATP level can be used as a metabolic biomarker for invasiveness and overproliferation cancer cell phenotype.Under this method, fluorescent ATP markers, such as vital dye BioTrackerTMATP-Red 1 (EMD Millipore Corporation, Burlington, Massachusetts) can be used for quantifying the mitochondrial ATP level in cancer cell group, and separate high ATP cancer cell subgroup and low ATP cancer cell subgroup by flow cytometry.The phenotypic analysis of these subgroups shows that high mitochondrial ATP is a metabolic feature that gives cancer cell overproliferation, dryness, non-anchorage dependency, antioxidant capacity and multidrug resistance.Similar results were obtained in number with four human breast cancer cell lines, i.e., MCF7, T47D, MDA-MB-231 and MDA-MB-468.

[0025] By combining ATP with other CSC markers such as CD44 or ALDH activity, the CSC population can be favorably classified into two subpopulations. The high CD44 / high ATP subpopulation has approximately twice the level of anchorage-independent growth as the high CD44 / low ATP subpopulation. Therefore, high CD44 / low ATP cancer cells represent a more dormant CSC population. Importantly, these results suggest that ATP levels may be a functional regulator of CSC dormancy.

[0026] The present method also includes complementary bioinformatics data, which show that mitochondrial ATP synthesis is involved in the stemness, metastasis and detection of circulating tumor cells (CTCs). The present invention discloses a five-element ATP-related metastasis gene signature comprising ABCA2, ATP5F1C, COX20, NDUFA2 and UQCRB. According to these clinical findings based on metastasis, high ATP MDA-MB-231 cells show a significant increase in the ability to undergo in vitro cell migration and invasion and spontaneous metastasis in vivo.

[0027] Therefore, the present method provides a new cellular platform for systematically identifying, studying and targeting stemness, multidrug resistance and metastasis of cancer cells.The present disclosure also mechanistically explains the positive therapeutic benefits of i) nutritional fasting and ii) caloric restriction mimetics in improving cancer treatment by inducing ATP depletion.

[0028] In an embodiment of the present method, the vital dye ATP-Red 1 is used as a molecular probe for identifying and separating high and low ATP subpopulations of cells (more specifically cancer cells and CSCs). The high ATP subpopulation of cancer cells is larger, more vigorous, overproliferates and undergoes anchorage-independent growth, which is consistent with a more "stem-like" phenotype. These ATP-rich cells can be targeted by ATP depletion therapy to eradicate CSCs that are "optimally surviving" in energy, reduce resistance and prevent metastasis.

[0029] Some embodiments of the present method can take the form of a purified composition of hyperproliferative cancer stem cells, which are in the form of a cell subpopulation from a human cancer cell population, which expresses a range of fluorescent signals in response to a fluorescent adenosine triphosphate (ATP) imaging probe, and the cell subpopulation expresses the upper part of the range of fluorescent signals based on ATP. The fluorescent ATP imaging probe can be, for example, BioTracker ATP-Red 1. Depending on the embodiment, the upper or high ATP subpopulation can be the top 10%, 5%, or 1% of the fluorescent signal based on ATP. Other parts can be used. In some embodiments, the composition is positive for the CD44 marker. In some embodiments, the composition is positive for the ALDH marker. In some embodiments, the composition is frozen.

[0030] In some embodiments, the method can take the form of a purified cell composition comprising a cancer stem cell subpopulation stained with a fluorescent ATP imaging probe and expressing a target portion of a range of ATP-based fluorescent signals of a cancer cell population. The cancer cell population expresses a range of ATP-based fluorescent signals, and the target portion of the range of ATP-based fluorescent signals can be the upper portion of the ATP-based fluorescent signal (e.g., a high ATP subpopulation) and / or the lower portion of the ATP-based fluorescent signal (e.g., a low ATP subpopulation). The target portion can be the bottom 10%, 5%, or 1% of the ATP-based fluorescent signal, or other selected portions.

[0031] Some embodiments may take the form of a purified composition of cells obtained by staining a population of human cancer cells with a fluorescent ATP imaging probe, isolating a portion of the population of human cancer cells having a target portion of an ATP-based fluorescent signal, and purifying the isolated cells. For example, the target portion may be the top 10% of the ATP-based fluorescent signal, the top 5% of the ATP-based fluorescent signal, the bottom 10% of the ATP-based fluorescent signal, the bottom 5% of the ATP-based fluorescent signal, etc. The isolated cells are positive for one of the CD44 marker and the ALDH marker.

[0032] Some embodiments may take the form of an ATP-based cell classification method. Cells in a cell population may be stained with a fluorescent ATP imaging probe that emits fluorescence when combined with ATP. ATP-based fluorescence signals of stained cells in a cell population may be measured. Stained cells may be separated based on a target portion of the ATP-based fluorescence signal. Fluorescence-activated cell sorting (FACS) and gating of a target portion of the ATP-based fluorescence signal may be used to separate stained cells. The gate may be set to collect the first 10% of the measured fluorescence signal and / or the last 10% of the measured fluorescence signal. It should be understood that other percentages may be used. The cell population may be derived from, for example, blood, urine, saliva, tumor tissue, noncancerous tissue, or metastatic lesions. Some embodiments may also include measuring the ALDH activity of separated cells, measuring the anchorage-independent growth of separated cells, measuring the mitochondrial mass of separated cells, measuring the glycolytic metabolism and oxidative mitochondrial metabolism of separated cells, measuring the cell cycle progression and proliferation rate of separated cells, and measuring the polyploidy of separated cells.

[0033] Embodiments of the present method can take the form of a method for separating and collecting metabolically active cells from a cell group. The cells in the cell group can be dyed with an ATP-labeled dye that emits fluorescence when combined with ATP. The fluorescent signal of the dyed cells can be measured in the cell group, and then the dyed cells are measured based on the measured fluorescent signal. Then, the cells separated by at least a portion of the fluorescent signal having the measured fluorescence signal being higher than a predetermined threshold and lower than one of the predetermined threshold can be collected, for example, by using a FACS instrument. The predetermined threshold includes a portion of the top of the measured fluorescence signal, such as the first 25%, the first 20%, the first 15%, the first 10%, the first 5%, the first 2% and the first 1%. Without departing from the present method, other percentages can be used. In some embodiments, the separated cells can be further separated based on a second marker, such as CD44(+), CD133(+), ESA(+), ALDEFLOUR(+), MitoTracker-High, EpCAM(+), CD90(+), CD34(+), CD29(+), CD73(+), CD90(+), CD105(+), CD106(+), CD166(+), and Stro-1(+). Other markers can be used without departing from the present method. In some embodiments, the second marker can take the form of an antibody coated on a magnetic bead.

[0034] The method can also take the form of a method for identifying and treating cancer stem cells in a biological sample. A biological sample can be obtained from a patient, and then cells in the biological sample can be stained with an ATP-labeled dye, wherein the ATP-labeled dye emits fluorescence when bound to ATP. The fluorescence signal of the stained cells in the cell population can be measured and then compared with a predetermined threshold indicating the presence of cancer stem cells. If the measured fluorescence signal exceeds the predetermined threshold, an ATP depletion therapeutic agent can be administered to the patient. The ATP depletion therapeutic agent can be, for example, doxycycline, tigecycline, azithromycin, enbopirovinium, atovaquone, bedaquiline, niclosamide, irinotecan, actinonin, CAPE, berberine, brutieridin, melitidin, oligomycin, AR-C155858, mitoriboscin, mitoketoscin, mitoflavoscin, TPP-derivatives, dodecyl-TPP, 2-butene-1,4-bis-TPP, or a combination of doxycycline, azithromycin and ascorbic acid.

[0035] In some embodiments, the method can take the form of a method for testing the anti-cancer activity of a candidate compound. A population of cancer cells can be stained with an ATP-labeled dye, such as BioTracker ATP-Red 1, that fluoresces when bound to ATP. The ATP-based fluorescence signal of the stained cells can be measured, and the stained cells can be separated based on the target portion of the ATP-based fluorescence signal to prepare a hyperactive cancer cell subpopulation. The candidate compound can be applied to the hyperactive cancer cell subpopulation; the effect of the candidate compound on the hyperactive cancer cell subpopulation can be measured. The ATP-labeled dye can be BioTracker ATP-Red 1. The target portion of the ATP-based fluorescence signal can be, for example, the top 25%, the top 20%, the top 15%, the top 10%, the top 5%, the top 2%, and the top 1%. In some embodiments, the hyperactive cancer cell subpopulation is positive for one of the CD44 marker and the ALDH marker. Embodiments may also involve measuring ALDH activity of a hyperactive cancer cell subpopulation, measuring anchorage-independent growth of cells of a hyperactive cancer cell subpopulation, measuring mitochondrial mass of a hyperactive cancer cell subpopulation, measuring glycolytic metabolism and oxidative mitochondrial metabolism of a hyperactive cancer cell subpopulation, measuring cell cycle progression and proliferation rate of a hyperactive cancer cell subpopulation, and measuring polyploidy of a hyperactive cancer cell subpopulation.

[0036] The present method can also take the form of a method for diagnosing and preventing metastasis risk in a cancer patient. The expression level of the 5-gene signature of ABCA2, ATP5F1C, COX20, NDUFA2, and UQCRB in a cancer biological sample of the patient can be determined and then compared to the baseline expression level of ABCA2, ATP5F1C, COX20, NDUFA2, and UQCRB in a non-cancerous biological sample from the patient. If the detected expression level exceeds the baseline expression level, an ATP-depleting compound can be administered to the patient. The ATP depleting compound can be, for example, doxycycline, tigecycline, azithromycin, enbopirovinium, atovaquone, bedaquiline, niclosamide, irinotecan, actinonin, CAPE, berberine, brutieridine, melitidin, oligomycin, AR-C155858, mitoriboscin, mitoketoscin, mitoflavoscin, TPP-derivatives, dodecyl-TPP, 2-butene-1,4-bis-TPP, or a combination of doxycycline, azithromycin and ascorbic acid.

[0037] Some embodiments can take the form of a kit for identifying circulating tumor cells in a biological sample. The kit can include reagents for identifying up-regulation of ABCA2, ATP5F1C, COX20, NDUFA2, and UQCRB in a biological sample, such as antibodies against proteins encoded by these genes. For example, the kit can be used in a liquid biopsy procedure for detecting CTCs.

[0038] The present method can also take the form of a method for detecting circulating tumor cells (CTCs) in a biological sample. The expression levels of ABCA2, ATP5F1C, COX20, NDUFA2, and UQCRB in the biological sample can be determined, and then if the determined expression levels are upregulated relative to the control, the presence of CTCs is determined. The biological sample can be, for example, blood, urine, saliva, tumor tissue, noncancerous tissue, or metastatic lesions. The sample can be further processed using the methods described herein to separate high ATP cells.

[0039] These and other embodiments will be apparent to those of ordinary skill in the art in view of the present specification, the appended claims, and the application incorporated herein by reference.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1A Shown is a heat map of ATP-related genes whose transcription is upregulated in both 3D growth conditions (anchorage-independent and in vivo tumor conditions) relative to 2D adherent growth. Figure 1B and 1C Volcano plots for the GSE2034 and GSE59000GEO datasets are shown. Figure 1D A Venn diagram showing the intersection of two breast cancer metastasis GEO datasets (GSE2034 and GSE59000) is used to identify ATP-related genes that are highly upregulated in both datasets as prognostic biomarkers for metastasis.

[0042] Figure 2A-2N These are data graphs showing the positive correlation between APT5F1C and genes CDH1, ALDH2, SOX2, VIM, CD44, EPCAM, MKI67, RRP1B, CXCR4, VCAM1, CDK1, CDK2, CDK4, and CDK6, respectively. Figure 2O-2Q These are data graphs showing the positive correlation between APT5F1C and UQCRB, COX20, and NDUFA2, respectively.

[0043] Figure 3A Kaplan-Meier curves for ER(+), relapse-free survivors (N=3,082) are shown. Figure 3B Kaplan-Meier curves of ER(+), distant metastasis-free survivors (N=1,395) are shown. Figure 3C Kaplan-Meier curves for ER(+), node-negative, tamoxifen-treated, relapse-free survivors (N=471) are shown.

[0044] Figure 4A Heat map showing the ATP-ABC gene expression profile, Figure 4B A heat map of OXPHOS gene expression profiles is shown. Figure 4C is a Western blot analysis of high and low ATP subpopulations of MDA-MB-231 cells.

[0045] Figure 5A An embodiment of a metabolic fractionation procedure according to an embodiment of the present invention is illustrated. Figure 5B An example of metabolic fractionation of MCF7 cells using ATP-Red1 to separate high ATP (top 5%) and low ATP (bottom 5%) cell subpopulations is illustrated.

[0046] Fig. 6A and 6B Results from a continuous real-time assay system are shown for cell proliferation of three cell subpopulations (Low ATP 5%, Bulk 5%, High ATP 5%).

[0047] Fig. 7A is a bar graph showing changes in luminescence of cells in the high ATP MCF7 subpopulation, Figure 7B Results of mammosphere formation assays for the high-ATP, bulk, and low-ATP subpopulations are shown. Figure 7C Comparative images of cell subpopulations after the assay are shown. Fig.7DThe signal intensity of CD44 and ALDH positive subpopulations is shown. Fig. 7E Cell-Titer-Glo results for this analysis are shown.

[0048] Fig. 8A and 8B Results of metabolic profiling of 3D mammospheres and high-ATP MCF7 cells are shown.

[0049] Fig. 9A and 9B Cell-Titer-Glo and 3D mammosphere formation results using 10% gating for high and low ATP subpopulations of MCF7, T47D, MDA-MB-231, and MDA-MB-468 cells are shown.

[0050] Fig. 10A Luminescence of high ATP subpopulation and low ATP subpopulation (10%) in MCF7 cell population after 24 hours is shown. Figures 10B to 10E The results of metabolic flux analysis of high-ATP and low-ATP subpopulations are shown.

[0051] Figures 11A-11D Cell cycle progression of MCF7, T47D, MDA-MB-468, and MDA-MB-231 cells is shown, using FACS analysis with propidium iodide to detect DNA content.

[0052] Fig. 12A The results show the resistance of the low ATP subpopulation (bottom 5%). Fig. 12B The drug resistance results for the high ATP subpopulation (top 5%) are shown.

[0053] Fig.13A and 13B The results of mammosphere assay formation of double-labeled cells (CD44 and ATP) in MCF7 cells and MDA-MB-231 cells are shown, respectively. Fig. 13C and 13D The results of mammosphere assay formation of double labeled cells (ALDH activity and ATP) in MCF7 cells and MDA-MB-231 cells are shown, respectively.

[0054] Fig.14A and 14B Shown are the results of migration and invasion assays of the high ATP subpopulation of MDA-MB-231 cells.

[0055] Fig.15 Results of a spontaneous metastasis in vivo CAM assay are shown.

[0056] Figures 16A-16CLuminescence changes, cell cycle progression, and mammosphere formation assay results of Tempo-ATP MCF7 cells are shown, respectively.

[0057] describe

[0058] The following description illustrates embodiments of the method in sufficient detail to enable the practice of the method. Although the method is described with reference to these specific embodiments, it should be understood that the method can be embodied in different forms, and the description should not be interpreted as limiting any appended claims to the specific embodiments set forth herein. On the contrary, these embodiments are provided to make this disclosure thorough and complete and to fully convey the scope of the method to those skilled in the art.

[0059] This description uses various terms that should be understood by those of ordinary skill in the art. For the avoidance of doubt, the following clarification is made. The terms "treat", "treated", "treatming" and "treatment" include reducing or alleviating at least one symptom associated with or caused by the state, disorder or disease (particularly cancer) being treated. In certain embodiments, treatment includes reducing and / or alleviating at least one symptom associated with or caused by the cancer being treated by a compound of the present invention. In some embodiments, treatment includes causing a class of cells such as CSCs to die in a specific cancer in a host, and can be achieved by preventing further proliferation of cancer cells, and / or inhibiting CSC function by, for example, depriving these cells of energy generation mechanisms. For example, treatment can be to reduce one or more symptoms of cancer, or to completely eradicate cancer. As another example, the present method can be used to inhibit mitochondrial metabolism in cancer, eradicate (e.g., kill at a rate greater than the proliferation rate) CSCs in cancer, eradicate TICs in cancer, eradicate circulating tumor cells in cancer, inhibit proliferation of cancer, target and inhibit CSCs, target and inhibit TICs, target and inhibit circulating tumor cells, prevent metastasis (i.e., reduce the likelihood of metastasis), prevent recurrence, sensitize cancer to chemotherapeutic agents, sensitize cancer to radiation therapy, and sensitize cancer to phototherapy.

[0060] The terms "cancer stem cells" and "CSC" refer to a subpopulation of cancer cells within a tumor that have the ability to self-renew, differentiate, and tumorigenicity when transplanted into an animal host. Compared to "bulk" cancer cells, CSCs have increased mitochondrial mass, enhanced mitochondrial biogenesis, and higher activation of mitochondrial protein translation. As used herein, "circulating tumor cells" are cancer cells that have shed from a primary tumor into the vasculature or lymphatics and are carried around the body in the blood circulation. The CellSearch Circulating Tumor Cell Test can be used to detect circulating tumor cells.

[0061] The phrases "high ATP" and "low ATP" refer to cell subpopulations from a starting cell population with upper and lower ATP-based fluorescent signals representing ATP-based fluorescent signals, respectively. The upper portion may represent the first 25%, or the first 20%, or the first 15%, or the first 10%, or the first 5%, or the first 2%, or the first 1% of the fluorescent signal based on ATP of the starting cell population. The lower portion may represent the last 25%, or the last 20%, or the last 15%, or the last 10%, or the last 5%, or the last 2%, or the last 1% of the fluorescent signal based on ATP of the starting cell population.

[0062] As used herein, the phrase "pharmaceutically effective amount" means the amount that must be administered to a host or a cell, tissue or organ of a host in order to achieve a therapeutic result, such as regulating, modulating or inhibiting protein kinase activity, such as inhibiting protein kinase activity or treating cancer. A physician or veterinarian having ordinary skills in the art can easily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start with multiple doses of the compound of the invention used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0063] Bioinformatic analysis demonstrated a role for mitochondrial ATP synthesis in 3D anchorage-independent growth, stemness, and distal metastasis. In particular, using a bioinformatic approach, mitochondrial ATP synthesis was identified as a key determinant of 3D anchorage-independent growth and metastasis. Existing proteomic profiling data were queried to compare 2D monolayers versus 3D mammospheres in two different ER(+) breast cancer cell lines (MCF7 and T47D). Overall, 21 ATP-related proteins were found to be upregulated in 3D-mammospheres in both datasets from 1,519 common proteins in both cell lines. Table 1 below shows these proteins and accession numbers, as well as the fold change in expression in MCF7 and T47D cells (spheres versus 2-D adherent growth). Of these 21 ATP-related proteins, seven mitochondrial ATP synthase subunits were detected, including ATP5F1B, ATP5F1C, ATP5IF1, ATP5MG, ATP5PB, ATP5PD, and ATP5PO. Using Ingenuity Pathway Analysis (IPA) software, we observed that predicted upstream regulators of 3D anchorage-independent growth were highly conserved between the two cell lines and were particularly correlated with existing IPA datasets on tumor growth and tumor cell proliferation.

[0064]

[0065]

[0066] Table 1. 21 ATP-related proteins upregulated in 3D mammospheres of MCF7 and T47D cell lines.

[0067] We also reanalyzed the GEO transcriptional profiling dataset and compared 2D growth, 3D growth, and in vivo tumor growth of MDA-MB-231 cells (a triple-negative breast cancer cell line). Figure 1A A heat map of ATP-related genes is shown, whose transcription is upregulated relative to 2D adherent growth in both 3D growth conditions (anchor-independent conditions and in vivo tumor conditions). The first column identifies the gene, the second column shows the expression profile in 2D MDA-MB-231 cells, the third column shows the expression profile in 3DMDA-MB-231 cells, and the fourth column shows the expression profile in xenograft MDA-MB-231 cells. Darker cells represent fewer fold changes, and lighter cells represent higher fold changes. The heat map shows the logarithm of the fold change, for example, the shallowest cells are + / -4. In the 2D MDA-MB-231 column, lighter cells represent negative changes (for example, ATP11A-AS1 shows a -4log fold change), while lighter cells in the 3D and xenograft columns represent positive changes (for example, ATP12A shows a 4log fold change).

[0068] Transcriptional expression of ATP-related genes (OXPHOS and ATP-related transporters) in two different GEO datasets associated with human breast cancer metastasis can be used to identify ATP-related genes associated with metastasis. Figure 1B and 1C Volcano plots for the GSE2034 and GSE59000 GEO datasets are shown. Specifically, Figure 1B Comparison of gene expression in metastatic and non-metastatic scenarios (GSE2034), Figure 1C Gene expression in metastatic contexts was compared to that in primary tumors (GSE59000). Volcano plots were generated by examining annotations present in OncoLand Metastatic Cancer (QIAGEN OmicSoft Suite) and by performing a functional "core analysis" of genes annotated with an uncorrected p-value cutoff of less than 0.05 using Ingenuity Pathway Analysis software (IPA; QIAGEN). In both GEO datasets, transcriptional profiles of ATP-related genes (OXPHOS and ATP-related transporters) were increased and specifically associated with metastasis.

[0069] Figure 1D A Venn diagram showing the intersection of two breast cancer metastasis GEO datasets (GSE2034 and GSE59000) is used to identify ATP-related genes that are highly upregulated in both datasets as prognostic biomarkers for metastasis. Figure 1B and 1C As described, the intersection of the two GEO datasets was performed using IPA software. The overlapping set of 1,055 genes contained only five ATP-related genes. These ATP-related genes, namely ABCA2, ATP5F1C, COX20, NDUFA2, and UQCRB, were highly upregulated in both metastasis GEO datasets and therefore have prognostic value in predicting cancer metastasis. These five ATP-related genes can be used as an ATP-related metastasis gene signature, metastasis prognosis. Most notably, ATP5F1C (also known as ATP5C1) encodes the gamma subunit of the soluble F1 catalytic core of the mitochondrial ATP synthase. UQCRB is an essential component of mitochondrial complex III, where it functionally binds ubiquinone and is involved in electron transport. COX20 is a chaperone protein that is essential for the assembly of mitochondrial complex IV. NDUFA2 is essential for the function of mitochondrial complex I. Finally, ABCA2 is a member of the ATP-binding cassette transporter gene family.

[0070] In true breast cancer metastatic lesions, ATP5F1C transcriptional expression was positively correlated with the co-expression of: i) five metastasis marker genes (EPCAM, MKI67, RRP1B, VCAM1, CXCR4); ii) four cell cycle regulatory genes (CDK1, CDK2, CDK4, CDK6); and iii) 11 CSC marker genes (CDH1, ALDH2, ALDH1BA1, ALDH9A1, SOX2, VIM, CDH2, ALDH7A1, ALDH1B1, CD44, ALDH3B2, listed in order of statistical significance). Figure 2A-2N is a data graph showing that APT5F1C is positively correlated with each of these genes in the order CDH1, ALDH2, SOX2, VIM, CD44, EPCAM, MKI67, RRP1B, CXCR4, VCAM1, CDK1, CDK2, CDK4, and CDK6.

[0071] In addition, ATP5F1C transcript expression was also positively correlated with the co-expression of mitochondrial complex IV, mt-DNA-encoded transcripts, and the other three members of the quintuple transfer gene signature, namely UQCRB, COX20, and NDUFA2. Figure 2O-2Q Figure 2 is a data graph showing positive correlations between APT5F1C and UQCRB, COX20, and NDUFA2, respectively. The expression of two members of this transfer gene signature, ATP5F1C and UQCRB, is functionally related to the maximum oxygen uptake (V O2max ) and a high percentage of type 1 fibers (mitochondria-rich).

[0072] ATP5F1C expression in skeletal muscle also increased significantly after exercise training, reflecting the improved muscle fitness of patients. In contrast, ATP5F1C levels decrease with age and are reduced in patients with progeria syndrome. These results highly suggest that high ATP5F1C expression is a biomarker for increased mitochondrial ATP production at the cellular level.

[0073] Using Kaplan-Meier (KM) analysis, we identified ATP5F1C as a prognostic biomarker for distant metastasis and tumor recurrence, particularly in ER(+) patients who were node-negative at diagnosis and received tamoxifen (hazard ratio (recurrence-free survival) = 2.77; P = 3.4E-06; N = 471). Figure 3A Kaplan-Meier curves for ER(+), relapse-free survivors (N=3,082) are shown. Figure 3B Kaplan-Meier curves of ER(+), distant metastasis-free survivors (N=1,395) are shown. Figure 3CKaplan-Meier curves for ER(+), node-negative, tamoxifen-treated, relapse-free survivors (N=471) are shown.

[0074] Using histochemical activity staining for mitochondrial complex IV in surgically resected lymph nodes, these results are consistent with previous studies indicating that mitochondrial activity is functionally upregulated in breast cancer metastatic lesions. In addition, the inventors previously demonstrated that transcription of 16 members of the ATP5 gene family was upregulated in human breast cancer cells relative to adjacent stromal cells in samples derived from N=28 breast cancer patients, including ATP5F1C (4.64-fold; p=1.14E-05).

[0075] An existing GEO dataset (GSE55470) was used to evaluate the use of ATP-related genes and OXPHOS genes as transcriptional biomarkers of circulating tumor cells (CTCs) from breast cancer patients. Figure 4A A heat map of the ATP-ABC gene expression profile in this dataset is shown and a legend is included. Figure 4B based on Figure 4A The same legend in shows a heat map of the OXPHOS gene expression profile. In general, the lighter the cell, the higher the absolute value of expression. It is difficult to distinguish between positive and negative fold changes using black and white for this application. For control blood, most of the cells in the first five columns are green in the original heat map, indicating negative expression fold changes. Most of the cells in the remaining columns are red, indicating positive expression fold changes. Overall, this data suggests that high ATP content in CTCs can be used as a biomarker to identify and track CTCs in whole blood, potentially improving cancer diagnosis and preventing metastatic spread.

[0076] Figure 4C Western blot analysis results of the MDA-MB-231 cells of high ATP subgroup and low ATP subgroup are shown.Results show that mitochondrial markers and CTC markers are all upregulated in high ATP MDA-MB-231 cells.Relative to the MDA-MB-231 cells in low ATP subgroup, the mitochondrial markers of complex I to V, including ATP5F1C, are overexpressed in the MDA-MB-231 cells of high ATP subgroup.In addition, two known CTCs and transfer markers (VCAM-1 and Ep-CAM) are overexpressed in the MDA-MB-231 cells of high ATP subgroup.β-actin and β-tubulin are used as markers of equal protein load.

[0077] In summary, bioinformatics data and analysis show that increased mitochondrial ATP synthesis may be a key driver of 3D non-anchorage-dependent growth and metastasis. Based on this analysis, compared with cancer cells with lower ATP levels, cancer cells with the highest ATP levels will have high proliferative properties, more stemness, non-3D anchorage-dependent growth, and other invasive behaviors. Similarly, those cells with the lowest ATP levels will be more dormant. These two subpopulations are of considerable value for the purposes of cancer research and drug screening in particular. The present method provides a method for separating these subpopulations from cancer cell populations by metabolic fractionation. Cancer cell populations have many subpopulations. CSCs are small cancer cell subpopulations with self-renewal properties that can differentiate, and they show tumorigenicity when transplanted. However, as described herein, not all CSCs are created equal. CSCs separated and purified based on ATP levels have unique phenotypic properties that have not been found in naturally occurring cancer cell populations or even in CSCs separated and purified using conventional markers such as CD44, CD24, and CD133.

[0078] Under this method, cells, preferably cancer cells, can be graded based on metabolic conditions using fluorescent ATP-labeled dyes such as ATP-Red 1 and flow cytometry. ATP levels ultimately determine the phenotypic characteristics of cancer cells, such as "stemness" and proliferation capacity. Therefore, ATP-labeled dyes can be used to identify and purify energetically "optimally survivable" cancer cells from a total cell population. The inventors have selected Biotracker ATP-Red 1 (fluorescent vital dye) to label ATP in living cancer cells. It should be understood that other fluorescent ATP imaging probes, including probes developed later, can be used without departing from this method. Preferably, the fluorescent ATP imaging probe targets mitochondrial ATP.

[0079] ATP-Red 1 is not normally fluorescent, but fluoresces when bound to ATP (but not to any other related nucleotides or metabolites, including ADP). More specifically, BioTracker ATP-Red1 does not recognize sugars (arabinose, galactose, glucose, fructose, ribose, sorbose, sucrose, or xylose) or other nucleotides (AMP, ADP, CMP, CDP, CTP, UMP, UDP, UTP, GMP, GDP, or GTP). Importantly, this fluorescent ATP imaging probe exhibits an "on" fluorescence response to ATP, with a nearly 6-fold increase in fluorescence. Using fluorescence microscopy, ATP-Red 1 was primarily detected within mitochondria, which are the primary source of cellular ATP production. Therefore, ATP-Red1 is preferred as a fluorescent probe for metabolic fractionation of cancer cell populations by flow cytometry.

[0080] Cancer cell populations can be separated or graded into high ATP cell subpopulations and low ATP cell subpopulations, and then phenotypes are characterized. These subpopulations can be defined according to the percentage before and after the fluorescence signal (e.g., front and rear 20%, front and rear 1%, etc.), and according to the FACS gating cutoff value of cell selection and collection based on the selected percentage. The data disclosed herein mainly rely on the front and rear 5% and the front and rear 10% as the gating cutoff value, but it should be understood that other percentages can be used without departing from the method. Of course, the percentage should be less than 50%, and it should be expected that the larger the percentage, the less specific the phenotype characterization for a given cell population will be.

[0081] Figure 5A The embodiment of the metabolic fractionation procedure of the present embodiment is described. Fluorescent ATP imaging probe can be dissolved in culture medium and incubated 501. The results described herein relate to 5 μM Biotracker ATP-Red, which is dissolved in culture medium and incubated in cells for 30 minutes as a fluorescent ATP imaging probe. The cells are then washed with PBS and digested with trypsin, and resuspended in FACS buffer, and passed through a 40 μm cell filter 503. Cells derived from 3D spheroids or 2D adherent conditions are analyzed using a FACS sorter (e.g., SONY SH800) 505. Using ATP-based fluorescent signals (e.g., front / back 1%, 2%, 3%, 4%, 5%, 10%, etc.), cells are gated and sorted 507 with the desired ATP content. Figure 5B Illustrated using ATP-Red 1 that MCF7 cells are subjected to metabolic fractionation, to separate the example of high ATP (front 5%) and low ATP (back 5%) cell subpopulations. For comparison purposes, the main body (5%) group was also selected. The right figure shows the cell count under different fluorescence intensities, and identifies the region of the median of high ATP (front 5%) subpopulation, low ATP (back 5%) subpopulation and main body. Figure 5B The left panel shows the average ATP-based fluorescence signal for each subpopulation. Based on the average signal intensity, we estimated that the ATP level of the high-ATP MCF7 cells was approximately 15 times higher than that of the low-ATP population and approximately 2 times higher than that of the bulk cell population.

[0082] Fig. 6A and 6B Results from a continuous real-time assay system for cell proliferation for all three cell subpopulations (low ATP 5%, bulk 5%, high ATP 5%) are shown. The RTCA DP instrument was used to assess cell proliferation. Cells were first sorted for ATP content, counted, and plated (1x 10 4The 3 subpopulations (low ATP 5%, bulk 5%, high ATP 5%) were graphically represented. The results showed that after 120 hours, the proliferation capacity of the high ATP group was about 2 times that of the bulk cell group and about 5 times that of the low ATP group. Data represent mean ± SD, n = 3. One-way ANOVA, Dunnett's multiple comparison test, **p < 0.001, ***p < 0.001, ****p < 0.0001. From Fig. 6A As can be seen, the cell index of the high ATP subpopulation is significantly higher than that of the other subpopulations, and the cell index of the low ATP subpopulation is significantly lower than that of the other subpopulations. Figure 6B The slope of the cell index over time for each subpopulation is shown. At each time point (24, 48, 72, 96 and 120 hours), the slope of the high ATP cell population was significantly higher than that of the other two subpopulations. Data represent mean ± SD, n = 3. Two-way ANOVA, corrected by Tukey, *p < 0.01. Obviously, the proliferation rate of the high ATP subpopulation was the highest throughout the 120-hour evaluation. The results show that the proliferation capacity of the high ATP population is at least about 2 times that of the bulk cell population and at least about 5 times that of the low ATP population. This indicates that mitochondrial ATP levels are a key determinant of MCF7 cell proliferation, and that this method of metabolic fractionation using fluorescent ATP imaging probes is an effective technique for identifying the cell subpopulations with the strongest and lowest proliferation capacity.

[0083] Further assays confirmed that the high ATP MCF7 subpopulation was energetically hyperproliferative with significantly increased 3D anchorage-independent growth, cancer stem cell markers, and mitochondrial mass. To confirm the selectivity of ATP-Red 1, ATP levels in cells were measured using Cell-Titer-Glo following flow cytometry. However, since Cell-Titer-Glo is a luciferase-based assay, it requires cell lysis to detect ATP levels and therefore cannot be used for live cell sorting or imaging. After cell counting, the cells were then analyzed using Varioskan TM LUX plate reader, using equal numbers of single cells to assess their relative ATP content by luminescence. Fig. 7A The bar graph shows that the ATP level of cells in the high ATP MCF7 subpopulation increased by at least 15 times that of the low ATP cell population, while the ATP increase of the bulk cells was about 7 times that of the low ATP cell population. This also shows that the ATP level of the high ATP subpopulation in the MCF7 population is at least twice that of the bulk cells.

[0084] A 3D mammosphere assay was used to measure anchorage-independent growth, a functional readout of CSC activity and CSC proliferation. Figure 7BShown are the results of 3D mammosphere assays of high ATP subpopulation, bulk subpopulation, and low ATP subpopulation using 5% as a gating cutoff. Figure 7C Comparative images of these cell subpopulations after assay are shown. 3D mammosphere images were acquired using an EVOS FL Auto2 microscope. Panels represent cells from the 3 sorted cell populations. Representative images are shown. A 4X objective was used. Scale bar = 1,000 μm. The high ATP MCF7 cell subpopulation had a 9-fold increase in 3D sphere formation compared to the low ATP subpopulation and almost double the mammosphere formation of the bulk subpopulation. These data suggest that high ATP cells are capable of better 3D anchorage-independent growth than the bulk CSC population.

[0085] Two well-established CSC markers, CD44 and ALDH activity, were used to examine the “stemness” of the subpopulations. Fig.7D It is shown that when a FACS gating cutoff of 5% is used, the high ATP MCF7 cell subpopulation (right bar) is almost 4 times more enriched in CD44 cell surface expression than the low ATP subpopulation (left bar), and the enrichment in ALDH activity is about 5.5 times that of the low ATP subpopulation. Similar results were obtained using MitoTracker-Deep-Red, a well-established mitochondrial mass marker, which revealed that the increase in the high ATP MCF7 subpopulation was 3 times that of the low ATP subpopulation. Mitochondrial mass is a specific marker of CSC stemness.

[0086] These indicate that the metabolic fractionation of this method enriches CSC activity in high ATP subpopulations. Importantly, high ALDH activity is considered a biomarker for EMT (epithelial-mesenchymal transition) in CSCs, while CD44 is considered more like an epithelial CSC marker. Therefore, epithelial and mesenchymal CSCs are greatly enriched in high ATP cell subpopulations.

[0087] High ATP MCF7 cell populations were also selected using fluorescent activity probes targeting antioxidant capacity and pluripotency. The effectiveness of the BioTracker ATP-Red 1 imaging probe was compared with several other fluorescent vital dyes, particularly for selectivity against high ATP cell populations. To this end, MCF7 cell 2D monolayers were harvested with trypsin and vitally stained with a panel of 5 other fluorescent BioTracker probes for i) antioxidant capacity, including cystine uptake (“Cysteine-FITC”) and gamma-glutamyl transpeptidase activity, or GGT; ii) pluripotency; iii) hypoxia; and iv) senescence (β-galactosidase activity, or β-Gal). Total ATP levels were then measured using Cell-Titer-Glo immediately following flow cytometry.

[0088] Fig. 7EThe Cell-Titer-Glo results for this analysis are shown, showing the luminescence fold change of the highest 5% (right bar for each probe) relative to the lowest 5% (left bar for each probe). It is worth noting that probes for antioxidant capacity (cystine uptake and GGT activity) and pluripotency were selected for high ATP subpopulations of MCF7 cells. However, among the other fluorescent active probes tested, the BioTracker probe that directly measures cystine-FITC uptake was the most effective in selecting high ATP cell subpopulations, but not as effective as ATP-Red 1 (3 times compared to 20 times). Interestingly, high antioxidant capacity is known to be strictly associated with stemness and drug resistance phenotypes. Hypoxia probes also favorably select high ATP cell subpopulations. This may be due to the association between hypoxia and increased mitochondrial biogenesis. However, the aging probe (β-galactosidase activity) did not select high ATP cell populations or low ATP cell populations.

[0089] Fig. 8A and 8B Results related to metabolic profiling of 3D mammospheres and high ATP MCF7 cells are shown. Intracellular ATP levels of MCF7 cells cultured as 2D monolayers or 3D spheroids were compared to better understand the metabolism underpinning 3D anchorage-independent growth. The latter cell population is known to be highly enriched for CSCs. Metabolite levels of MCF7 cells grown as 2D adherent monolayers or 3D mammospheres were compared using Promega kits (Cell-Titer-Glo, GSH / GSSG-Glo, NADP-NADPH-Glo, NAD-NADH-Glo). 2D monolayers and 3D mammospheres were first dissociated into single cells using trypsin, injected using a 25-gauge needle, and passed through a 40 μm cell strainer. After cell counting, equal numbers of single cells were then used to assess their relative luminescent content. Note that cells derived from 3D mammospheres had more than a 2-fold increase in ATP levels; nearly a 2-fold increase in reduced glutathione levels; more than a 2-fold increase in NADP-NADPH levels, and nearly a 1.5-fold increase in NAD-NADH levels, all relative to 2D monolayers. Data represent mean fold increase ± SD relative to adherent cells, n = 4. Unpaired t-test, **p < 0.005, ***p < 0.0005, ****p < 0.0001.

[0090] Fig. 8AThe luminescence changes of 3D spheroids (right bars) relative to 2D monolayers (adherent, left bars) for probes targeting ATP, GSH / GSSG, NADP-NADPH, and NAD-NADH were compared. Quantitative analysis of MCF7 cells derived from 3D spheroids showed a 2.3-fold increase in ATP levels compared to 2D monolayer cells. An approximately 2-fold increase in the GSH / GSSG ratio and NADP / H levels was observed, and similar results were obtained with NAD / H. These data are consistent with the idea that 3D anchorage-independent growth may also require increased antioxidant capacity.

[0091] Given the above, it is expected that the high ATP subpopulation of 2D monolayer cells has the ability to undergo 3D anchorage-independent growth. Under low-attachment conditions, >90% of MCF7 cells typically undergo anoikis, a specialized form of apoptotic cell death. Higher ATP levels may allow CSCs to better withstand the high stress of growth in suspension caused by the lack of cell-matrix attachment. However, higher energy reserves may also confer resistance to multiple stressors, leading to multidrug resistance.

[0092] Metabolic profiling of NAD / H and two key antioxidants, GSH and NADP / H, was performed in high-ATP and low-APT MCF7 cells using Promega kits (Cell-Titer-Glo, GSH / GSSG-Glo, NADP-NADPH-Glo, NAD-NADH-Glo). 2D monolayer cells were first stained with BioTracker ATP-Red 1 and sorted by ATP content by flow cytometry. After cell counting, equal numbers of single cells were then used to assess their relative luminescent content. Note that high-ATP cells showed a nearly 25-fold increase in ATP levels; a 6-fold increase in reduced glutathione levels; a nearly 8-fold increase in NADP-NADPH levels, and a >2-fold increase in NAD-NADH levels, all relative to low-ATP MCF7 cells. Data represent mean fold increase ± SD relative to low-ATP 5% cells, n = 4. Unpaired t-test, **p<0.005, ***p<0.0005. As Figure 8B As shown, the cells in the high ATP subgroup contain more than 1.5 times of NAD / H, more than 7.5 times of NADP / H, and more than 7 times of reduced glutathione (GSH / GSSG ratio), all relative to the cells in the low ATP subgroup. These data show that the MCF7 cells in the high ATP subgroup are more vigorous, and therefore, they enhance their antioxidant capacity. High levels of antioxidants are known to be associated with drug resistance in cancer cells, indicating a multidrug resistance phenotype. Therefore, the cells in the high ATP MCF7 subgroup simulate 3D metabolic phenotypes, which shows that the present method for separating high ATP cells from a population produces a unique phenotype, thereby having many potential uses.

[0093] High-ATP and low-ATP subpopulation phenotypes exist in multiple cancer types. High-ATP subpopulations of MCF7, T47D, MDA-MB-231, and MDA-MB-468 cells all exhibited increased 3D anchorage-independent growth. Compositions of high-ATP and low-ATP cell subpopulations from three other human breast cancer cell lines, T47D, MDA-MB-231, and MDA-MB-468 cells, were prepared with a FACS gating cutoff of 10%. The relative amounts of ATP in the high-ATP and low-ATP cell subpopulations were independently verified using Cell-Titer-Glo. 2D monolayers were first stained with BioTracker ATP-Red 1 and sorted by ATP content using flow cytometry. After cell counting, an equal number of single cells were then used to assess their relative luminescent content. In this series of experiments, we used a 10% cutoff to demarcate high-ATP and low-ATP cell populations. Note that this metabolic fractionation scheme can be successfully applied to other breast cancer cell lines. Data represent mean fold increase ± SD relative to 10% low ATP cells, n = 3. Unpaired t-test, *p < 0.05, **p < 0.005, ***p < 0.0005.

[0094] Fig. 9A and 9B Cell-Titer-Glo and 3D mammosphere formation results using high and low ATP subpopulations of 10% gated MCF7, T47D, MDA-MB-231, and MDA-MB-468 cells are shown. Fig. 9A It was shown that the high ATP subpopulation of all these cell lines exhibited the increase in ATP that characterizes the high ATP subpopulation phenotype, as demonstrated by a 2-3 fold increase in total ATP levels using the luciferase-based Cell-Titer-Glo assay. Fig. 9B As shown, similar results were obtained using the 3D spheroid assay, indicating a 1.75-fold to 3-fold increase in CSC activity and proliferation, depending on the cell line examined. Monolayer cells were first stained with BioTracker ATP-Red 1 and sorted by ATP content using flow cytometry. After cell counting, 5x10 3 Cells were seeded onto 6-well plates coated with poly-HEMA and counted after 5 days. Note that the high ATP populations of MCF7, T47D, MDA-MB-231, and MDA-MB-468 cells all showed increased 3D anchorage-independent growth capacity. Data represent mean fold increase ± SD relative to low ATP 10% cells, n = 3. Unpaired t-test, ***p < 0.0005, ****p < 0.0001.

[0095] Cells in the high-ATP subpopulation exhibited increases in oxidative mitochondrial metabolism, glycolytic rate, and cell cycle progression. Fig. 10A The luminescence of the high ATP subpopulation and the low ATP subpopulation (10%) in the MCF7 cell population after 24 hours is shown. After cell counting, the same number of single cells were used to analyze the luminescence of the MCF7 cell population 24 hours after plating using Varioskan TM The LUX plate reader assesses relative ATP content by luminescence. For these experiments requiring large numbers of cells, a 10% cutoff was used to define high and low ATP cell populations. Data represent mean fold increase ± SD relative to low ATP 10% cells, n = 3. Unpaired t-test, ****p < 0.0001. 24 hours after plating high ATP cells as a 2D monolayer, the observed increase in ATP levels was reduced by 3-fold, indicating that the high-viability, high-ATP phenotype is relatively transient, consistent with a more stem-like phenotype.

[0096] Figures 10B to 10E The results of metabolic flux analysis of high ATP subpopulation and low ATP subpopulation are shown. OCR (oxygen consumption rate) was determined by metabolic flux analysis using SeahorseXFe96. Please note that high ATP MCF7 cell groups show an increase in basal respiration and maximum respiration as well as mitochondrial ATP production. 24 hours after plating, the cell population was analyzed. The data represent the % fold increase ± SD relative to low ATP 10% cells, n = 3. Unpaired t test, *p < 0.05, **p < 0.005. ECAR (extracellular acidification rate) was determined by metabolic flux analysis using SeahorseXFe96. Please note that high ATP MCF7 cell groups show an increase in glycolysis. 24 hours after plating, the cell population was analyzed. The data represent the % average fold increase ± SD relative to low ATP 10% cells, n = 3. Unpaired t test, ns = not significant, ***p < 0.0005. Fig. 10B and 10C The energy profile in the Figure 2 shows that the high ATP subpopulation is metabolically more active relative to the low ATP population. 24 hours after cell attachment, the high ATP MCF7 cell monolayer exhibited a 2-fold increase in basal respiration, a 1.5-fold increase in maximal respiration, and a 3-fold increase in ATP production. Similarly, the high ATP MCF7 cell monolayer also exhibited a 1.5-fold increase in basal glycolytic rate. Fig. 10D and 10E The glycolytic rates in the ATP subpopulation indicated that the high ATP subpopulation had significantly higher bioenergetic potential than the low ATP subpopulation.

[0097] Evaluation of the proliferation capacity of the high ATP cell subpopulation showed that the high ATP cell subpopulation had significantly greater proliferation capacity than the low ATP subpopulation in multiple cancer types. DNA content was detected using FACS analysis using propidium iodide. Figures 11A-11DThe cell cycle progression in MCF7, T47D, MDA-MB-468 and MDA-MB-231 cells is shown. It can be seen that the proliferation capacity of the high ATP cell subgroup is significantly stronger than the low ATP cell subgroup according to the transition from the G0 / G1 phase to the S phase and the G2 / M phase. More specifically, the G0 / G1 phase is reduced from about 80-88% to 60-64%, while the S phase is increased from 4-8% to 9-21%. Similarly, the G2 / M phase is increased from 7-12% to 17-30%. These obvious increases in the cell cycle progression of the high ATP cell subgroup relative to the low ATP subgroup have been observed in all 4 cell lines. Overall, this means that in the 4 cell lines tested, the number of cells in the S phase increases to 1.9-3.6 times, and the number of cells in the G2 / M phase increases to 1.8-3.8 times. Interestingly, the greatest increase was observed in the S phase in MCF7 cells, whereas the greatest increase was observed in the G2 / M phase in MDA-MB-231 cells.

[0098] In contrast, the low-ATP population in each cell line was essentially quiescent, with 80-88% of cells in the G0 / G1 phase of the cell cycle, displaying the primary phenotype of cell cycle arrest. Thus, the low-ATP cell subpopulation fits well with the current definition of cancer cell dormancy.

[0099] Therefore, high ATP levels are a major determinant of "stemness" characteristics, anchorage-independent growth and cell proliferation. Therefore, the practical methods described herein allow the successful isolation of the bioenergetically "fittest" and most proliferative cancer cells from the total cell population and the formation of new compositions of cells with unique phenotypic properties. These properties have an impact on drug resistance.

[0100] MCF7 cells in the high ATP subpopulation exhibit a multidrug resistance phenotype. The differential sensitivity of high ATP and low ATP MCF7 cell subpopulations to four different classes of drugs was explored using a 3D-mammosphere assay, used as a functional readout of resistance. The drug classes included tamoxifen, doxycycline, DPI, and palbociclib. Fig. 12A Results for the low ATP subpopulation (bottom 5%) are shown. Fig. 12B Results for the high ATP subpopulation (top 5%) are shown. Fig. 12A and 12B Two concentrations of each drug class are shown.

[0101] Tamoxifen is an FDA-approved drug routinely used in the clinic to target ER(+) breast cancer cells, which often leads to tamoxifen resistance and treatment failure, resulting in tumor recurrence and distant metastasis. Interestingly, 3D mammosphere formation by low-ATP MCF7 cells was very sensitive to tamoxifen treatment, resulting in a reduction of approximately 40% at 1 μM and >90% at 5 μM. In contrast, Fig. 12B 3D-mammosphere formation of high ATP MCF7 cells was shown to be significantly resistant to tamoxifen, as 3D mammosphere formation remained high at 5 μM, accounting for more than 80% of vehicle-treated control levels. Thus, high ATP MCF7 cells were clearly resistant to tamoxifen.

[0102] Fig. 12B High ATP MCF7 cells were shown to be resistant to the mitochondrial OXPHOS inhibitor, diphenyleneiodonium (DPI). For example, DPI treatment of low ATP cells reduced 3D mammosphere formation by >90% at 100 nM. On the other hand, DPI treatment (100 nM) of high ATP cells only reduced 3D mammosphere formation by about 55%. Therefore, both subpopulations are sensitive to mitochondrial inhibitors, but high ATP cells are clearly more resistant.

[0103] Doxycycline is an FDA-approved antibiotic that acts as an inhibitor of mitochondrial ribosomal translation. Fig. 12A and Fig. 12B It was shown that at concentrations that are very effective against low ATP MCF7 cells, namely 25 μM and 50 μM, the high ATP subpopulation was largely resistant to doxycycline.

[0104] Palbociclib is an FDA-approved CDK4 / 6 inhibitor that is effective in Fig. 12A and 12B This is also evident in the results of the experiments. Palbociclib treatment of low ATP cells reduced the formation of 3D mammospheres by about 75% at 12.5 nM. However, palbociclib treatment of high ATP cells (12.5 nM) only reduced 3D sphere formation by about 50%. Therefore, high ATP cells are also more resistant to CDK4 / 6 inhibitors. It can be seen that the high ATP subpopulation is a phenotype that is resistant to several drug types.

[0105] Biotracker-ATP-Red 1 was compared to the well-established markers of stemness, CD44 and ALDH activity. To directly compare the effectiveness of ATP-Red 1 with other CSC markers, a dual labeling strategy was applied to both MCF7 and MDA-MB-231 cells. Cells were dual labeled with CD44 and ATP, and detected using different fluorescence channels. In the case of CD44 and ATP, this resulted in 4 experimental groups: high CD44 / high ATP, high CD44 / low ATP, low CD44 / high ATP, and low CD44 / low ATP.

[0106] After cell sorting, the four resulting subpopulations were subjected to 3D mammosphere assays as a functional readout of stemness. ATP and CD44 cell surface expression. Using MCF7 and MDA-MB-231 lines, 3D anchorage-independent growth was measured in different cell subpopulations after cell sorting as a functional readout of stemness. Briefly, 2D monolayers were first co-stained with BioTracker-ATP (PE channel) and anti-CD44 (APC channel) and subjected to flow cytometry using a SONY SH800 cell sorter. After cell counting, 5x10 3 Cells were seeded in 6-well plates coated with poly-HEMA, and 3D mammospheres were counted 5 days after plating. Fig.13A and 13B As shown, low CD44 / low ATP cells displayed the least anchorage-independent growth, as expected given the phenotypic properties of these subsets. Therefore, low CD44 / low ATP- cells were chosen as the point for normalization. Both cell subsets displayed the most anchorage-independent growth: high CD44 / high ATP and low CD44 / high ATP. Thus, in both MCF7 and MDA-MB-231 cells, high ATP levels are the major determinant of stemness compared to CD44.

[0107] Considering only the high CD44 population and double labeling with ATP allows the high CD44 population to be divided into 2 subpopulations, one with high proliferation capacity (high CD44 / high ATP) and one with low proliferation capacity (high CD44 / low ATP). Therefore, the high CD44 / low ATP population clearly exhibits significantly less anchorage-independent growth and represents a more "dormant" CD44(+)CSC subpopulation.

[0108] Almost identical results were obtained by double labeling with ADLH activity and ATP. After cell sorting, 3D anchorage-independent growth was measured in different cell subsets using MCF7 and MDA-MB-231 cell lines as a functional readout of stemness. Briefly, 2D monolayers were first co-stained with BioTracker-ATP (PE channel) and ALDH activity (APC channel) and subjected to flow cytometry using a SONY SH800 cell sorter. After cell counting, 5x10 3 Cells were seeded in 6-well plates coated with poly-HEMA and 3D mammospheres were counted 5 days after plating. Fig. 13C and 13DResults of mammosphere formation assays for MCF7 and MDA-MB-231 cell lines double labeled for ALDH activity and ATP are shown, respectively. As expected, the two cell populations that exhibited the least anchorage-independent growth were high ALDH / high ATP and low ALDH / high ATP. Thus, in both MCF7 and MDA-MB-231 cells, high ATP levels are the main determinant of stemness compared to ALDH. Likewise, double labeling with ATP allowed the high ALDH population to be separated into 2 subpopulations, one with high proliferative capacity (high ALDH / high ATP) and one with low proliferative capacity (high ALDH / low ATP).

[0109] The above demonstrates that the present method is a powerful and effective method that uses ATP as a second marker of dormancy to subdivide CSCs into a more active, hyperproliferative subpopulation and a more dormant subpopulation. The results also indicate that ATP levels are a functional regulator of CSC dormancy.

[0110] The role of mitochondrial ATP in cell migration, invasion and spontaneous metastasis was also explored. The data show that mitochondrial ATP is an energy biomarker for the metastatic process of cancer cells. MDA-MB-231 cells are a recognized model for studying cell motility and metastasis in vitro and in vivo. Using Transwells, the ability of high and low ATP subpopulations of MDA-MB-231 cells to perform cell migration and invasion was assessed by using a modified Boyden chamber assay. The bulk (5%) group was also selected for comparison. In order to study invasion, Transwells were coated with an extracellular matrix, i.e., Matrigel, to prevent simple cell migration. For cell migration and invasion assays, serum was used as a chemical attractant. Crystal violet staining intensity and cell number were used to independently quantify migration and invasion parameters.

[0111] Fig.14A and 14B The results of the migration and invasion analysis are shown. Compared with low ATP cells, the ability of high ATP MDA-MB-231 cells to carry out cell migration is improved by 20-40 times. As expected, body (5%) cells show an intermediate phenotype. Compared with low ATP cell populations, the ability of high ATP MDA-MB-231 cells to carry out invasion is improved by 15-25 times. Therefore, high ATP MDA-MB-231 cells represent pro-metastatic cell subpopulations in vivo.

[0112] For further evaluation, spontaneous transfer was quantitatively measured using recognized in vivo transfer assay (involving the chorioallantoic membrane (CAM) of chicken eggs). After cell sorting to separate high ATP cell subpopulation and low ATP cell subpopulation, 30,000 cells (MDA-MB-231) of inoculum were added to the CAM of each egg (E9 day), and then the eggs were randomly grouped. At E18 day, the CAM below was collected to use the specific primers for people Alu sequences to evaluate the number of transferred cells by qPCR analysis. Uninjected eggs were also evaluated in parallel, as specific negative controls. More than 20 eggs were processed in each experimental condition.

[0113] Fig.15 The results of the spontaneous metastasis in vivo CAM assay are shown. The data show that the MDA-MB-231 cells in the high ATP subpopulation are 4.5 times more metastatic than the low ATP cell subpopulation. These subpopulations are derived from the same cell line. Therefore, the MDA-MB-231 cells in the high ATP subpopulation represent a pro-metastatic CSC subpopulation. As described above in conjunction with Figure 4C As discussed, MDA-MB-231 cells in the high ATP subpopulation also overexpressed two CTC and metastatic markers (VCAM-1 and Ep-CAM), suggesting that hyperproliferative CSCs are the CTCs responsible for seeding distant metastases.

[0114] Therefore, the present method can be used to detect the possibility of cancer metastasis. For example, biological samples from cancer can be metabolically graded to assess the content of high ATP subpopulations, and this content can be used to assess the possibility of cancer metastasis. Early detection and analysis of cells in high ATP subpopulations of cancer patients provide valuable opportunities for diagnosing metastasis risk and determining appropriate treatment (e.g., treating with ATP depletion therapeutic agents discussed herein).

[0115] Tempo-ATP protein biosensor purified from high-ATP MCF7 cells provides independent validation of the use of ATP as a new biomarker for cancer cell stemness. Tempo-ATP is a fluorescent protein biosensor, a completely different probe for detecting ATP levels in living cells, and was used to detect both high and low levels of ATP.

[0116] Tempo-ATP-MCF7 cells recombinantly overexpressing the cytoplasmic fluorescent protein ATP biosensor were custom generated by Tempo-Bioscience, Inc. (San Francisco, CA, USA) using a puromycin resistance marker for cell selection. This protein-based fluorescent ATP biosensor has an excitation wavelength of 517-519-nm and an emission wavelength of 535-nm. It consists of an ATP-binding peptide fused in frame with a GFP-like fluorescent reporter protein. Tempo-ATP-MCF7 cells were sorted for GFP content as a surrogate marker for cytoplasmic ATP content using a flow cytometer (excitation = 517-519-nm; emission = 535-nm). After cell counting, an equal number of single cells were used to assess their metabolic and phenotypic properties.

[0117] The results are as follows Figures 16A-16C The relative increase in luminescence of the high GFP subpopulation relative to the low GFP subpopulation is shown in Fig.16A Cell cycle progression data are summarized in Fig. 16B , and the results of the mammosphere formation assay are shown in Fig. 16C As expected from the discussion thus far, high-ATP Tempo-MCF7 cells exhibited a significant increase in ATP, more reduced glutathione, NADP / H, and NAD / H, as well as increased cell cycle progression and 3D anchorage-independent growth. The Tempo-ATP data independently validated the BioTracker ATP-Red 1 results, specifically, that high ATP levels are a key determinant of antioxidant capacity, cell proliferation, and 3D anchorage-independent growth. Although Tempo-ATP is potent, BioTracker ATP-Red 1 is significantly more potent because it localizes directly within mitochondria, the major cellular source of ATP production.

[0118] This approach demonstrates that high ATP production is a key driver of cancer cell "stemness" traits and proliferation. The observations disclosed herein may explain the molecular basis of metabolic heterogeneity observed in cancer cell populations, and its relationship to phenotypic behaviors such as i) rapid cell cycle progression and ii) anchorage-independent growth, both of which are required for CSC metastatic dissemination in vivo.

[0119] As demonstrated, ATP can be used as a biomarker to metabolically grade cancer cell populations and identify hyper-prolific and dormant subpopulations. This in turn suggests that ATP depletion therapy may be effective for treating hyper-prolific subpopulations and reduce or eliminate the possibility of tumor recurrence and metastasis.

[0120] Under the present method, important fluorescent dyes that allow technicians to measure ATP levels in living cells, such as BioTracker ATP-Red 1, can be used as imaging probes for metabolic fractionation. More particularly, BioTracker ATP-Red 1 staining can be combined with a bioenergetic fractionation scheme in which flow cytometry is performed on the total cell population to separate high ATP subpopulations and low ATP subpopulations of the population. MCF7 cells, an ER(+) human breast cancer cell line, have been used in many of the examples discussed above, but it should be understood that the present method can be used for any cell line and any cancer type. The metabolic fractionation method allows the most "viable" cancer cells to be isolated from the total cell population. Advantageously, the resulting high ATP cancer cell subpopulation can be targeted for eradication through ATP depletion therapy and serve as the basis for drug discovery and development. Given the phenotypic properties, the high ATP subpopulation can also be used to evaluate therapies that prevent or reduce the likelihood of recurrence and metastasis.

[0121] In parallel studies, the inventors identified more than 20 mitochondrial targeted therapeutics that can be used to effectively achieve ATP depletion therapy. These potential therapeutics include: FDA-approved drugs (doxycycline, tigecycline, azithromycin, embopirovinium, atovaquone, bedaquiline, niclosamide, irinotecan); natural products / nutraceuticals (actinonin, CAPE, berberine, brutieridine, melitidin); and experimental compounds (oligomycin, AR-C155858, Mitoriboscins (see International Application No. PCT / US2018 / 022403, which was filed on March 14, 2018 and is incorporated by reference in its entirety), Mitoketoscins (see International Application No. PCT / US2018 / 039354, which was filed on 2 The invention relates to the use of a combination of two antibiotics with vitamin C (doxycycline, azithromycin, and ascorbic acid) to target mitochondria, induce ATP, and induce mitochondria-derived ... Depletion and inhibition of CSC proliferation are particularly effective (see International Patent Application PCT / US2019 / 066541, filed on December 16, 2019, and incorporated by reference in its entirety). ATP depletion compounds can be existing compounds modified to increase efficacy, cell membrane penetration, and / or mitochondrial uptake, such as those described in International Patent Applications PCT / US2018 / 033466, filed on May 18, 2018, and PCT / US2018 / 062956, filed on November 29, 2018, and incorporated by reference in its entirety. For example, doxylidine conjugated to fatty acids (e.g., myristic acid) The compound can be used as an ATP depletion compound. In some cases, it may be appropriate to administer an increased dose of the compound, such as when the high ATP subpopulation shows resistance to the compound at the dose usually administered in the art. The compound can be administered. It should be understood that any of the above compounds can be used as an ATP depletion therapeutic agent that targets the high ATP subpopulation and prevents or reduces the possibility of recurrence and metastasis. It should also be understood that when the expression level of the 5-member gene signature of ABCA2, ATP5F1C, COX20, NDUFA2 and UQCRB in the patient's cancer biological sample is found to be increased relative to the patient's non-cancerous biological sample, any of the above compounds can be used as a therapeutic agent administered to the cancer patient.

[0122] Since many compounds are repurposed FDA-approved antibiotics with excellent safety, Phase II clinical trials are needed. For example, preliminary Phase II clinical studies of doxycycline have shown that this 50-year-old antibiotic is indeed effective in metabolically targeting the CSC population of patients with early breast cancer, as demonstrated by the use of CD44 and ALDH1 as specific CSC markers. It is expected that mitochondrial ATP depletion therapy functionally mimics fasting and / or caloric restriction, thereby more effectively starving CSCs. Under this method, fasting and / or caloric restriction can be included as part of anticancer therapy to increase the effectiveness of ATP depletion therapy. For example, patients receiving ATP depletion therapy can fast for a period of time, such as 12, 16, 24, 36 or 48 hours before receiving the administration of the therapeutic compound, and / or can fast for a period of time, such as 12, 16, 24, 36 or 48 hours after receiving the administration of the therapeutic compound. In some embodiments, fasting can be performed before and after the administration of the therapeutic compound to increase the ATP consumption effect. This is of great significance for preventing cancer and potentially extending human lifespan during aging.

[0123] Cells in the high ATP subpopulation displayed a multidrug resistance phenotype with enhanced antioxidant capacity. Previous studies have shown that high antioxidant capacity due to increased reduced glutathione levels, elevated NADPH, and activated NRF2 signaling significantly contributes to the development of multidrug resistance. MCF7 cells in the high ATP subpopulation have enhanced antioxidant capacity, increased reduced glutathione levels, and intrinsic resistance to four different classes of drugs (tamoxifen, palbociclib, doxycycline, and DPI). Therefore, the presence of a high ATP CSC phenotype may help to mechanistically explain the pathogenesis of multidrug resistance during cancer treatment. In this context, current cancer therapies may only allow metabolically "fittest" cancer cells to survive. These cells, in turn, pose the greatest risk of recurrence and metastasis.

[0124] The data disclosed above also show a direct causal relationship between mitochondrial "power" and tamoxifen resistance. For example, MCF7-TAMR cells with tamoxifen resistance generated by long-term exposure to increasing concentrations of tamoxifen show elevated levels of mitochondrial OXPHOS and ATP production. In MCF7-TAMR cells, acquired tamoxifen resistance is due to overexpression of two key antioxidant proteins (NQO1 and GCLC) and their active metabolic effects on mitochondrial metabolism, as revealed by unbiased proteomics analysis. In addition, recombinant overexpression of NQO1 or GCLC in MCF7 cells autonomously conferred an approximately 2-fold increase in mitochondrial ATP production and tamoxifen resistance. In addition, recombinant overexpression of somatic mutations (Y537S) in estrogen receptors (ER-α; ESR1) is clinically associated with acquired tamoxifen resistance in breast cancer patients, genetically conferring elevated mitochondrial biogenesis, OXPHOS, and high ATP production. MCF7-TAMR cells and MCF7-ESR1(Y537S) cells also showed considerable overlap in their proteomic profiles of functionally activated biological processes. Finally, 60 gene products functionally associated with mitochondrial ATP production predicted tamoxifen resistance in ER(+) / Luminal A breast cancer patients. These predictive biomarkers included 18 different mitochondrial ribosomal proteins (MRPs) and more than 20 different components of the mitochondrial OXPHOS complex. The data disclosed in this article indicate that "primitive" MCF7 cells in the high ATP subpopulation are intrinsically resistant to tamoxifen without previous exposure to tamoxifen. This has important clinical implications for optimizing the effectiveness of hormonal breast cancer therapy.

[0125] It has been reported that treatment with conventional chemotherapy regimens actually increases the number of CSCs while selectively killing "entity" cancer cells. Prior to this disclosure, no metabolic hypothesis was proposed to explain this phenomenon. Chan and colleagues (from Genentech, Inc.) examined the effects of gemcitabine and etoposide on total cancer cell populations. It is worth noting that they observed that after treatment with gemcitabine and etoposide, the surviving cell population showed increased ATP content, increased mitochondrial mass, and more mitochondrial respiration. However, they did not propose a mechanistic explanation for these observations, nor did they consider CSC populations. Instead, they simply concluded that measuring ATP is not a good way to evaluate the effectiveness of chemotherapeutic agents. In view of the data disclosed herein, another explanation for its results is that gemcitabine and etoposide selectively kill low ATP subpopulations and entity subpopulations of cancer cells, thereby enriching more stem-like and more drug-resistant, "viable" high ATP subpopulations. Therefore, new drug discovery that helps to eradicate high ATP subpopulations in cancer cells should be initiated.

[0126] Higher intracellular ATP levels have also been proposed to explain acquired resistance to oxaliplatin and cisplatin in various chronically treated colon and ovarian cancer cell lines (HT29, HCT116, A2780), although multiple mechanisms have been proposed, including increased glycolysis and / or mitochondrial metabolism. However, in previous studies, ATP levels were measured only after long-term selection of resistant cell populations. Therefore, a direct causal relationship between ATP production and resistance could not be established.

[0127] Previously, the inventors used a more indirect method to isolate "energetic" cancer stem cells (e-CSCS), which used autofluorescence to detect intracellular FAD, FMN and riboflavin content. See International Patent Application PCT / US2019 / 037860 filed on June 19, 2019, which is incorporated by reference in its entirety. However, the use of ATP-Red 1 is a direct method and a substantial improvement. For example, the use of high autofluorescence (AF; top 5%) to grade MCF7 2D monolayers resulted in a high AF cell population with a 1.5-fold increase in anchorage-independent growth and a nearly 2-fold increase in APT production. In contrast, in the present method, the use of ATP-Red 1 (top 5%) resulted in a high ATP MCF7 monolayer cell subpopulation with a 9-fold increase in anchorage-independent growth and a more than 15-fold increase in ATP content. High ATP subpopulations from other cancer cell lines (T47D, MDA-MB-231, and MDA-MB-468) exhibited similar hyperproliferative characteristics. Therefore, the use of ATP as a direct energy biomarker is far superior to autofluorescence. In addition, ATP-Red 1 can also effectively metabolically fractionate the other three breast cancer cell lines tested.

[0128] According to the traditional view of tumor dormancy, dormant cancer cells undergo a slower rate of cell proliferation and / or cell cycle arrest (quiescence) to avoid treatment-induced cell death, leading to multidrug resistance. The data disclosed in this article show the opposite phenomenon: using 3D mammosphere assay as a readout, MCF7 cells in the low ATP subpopulation proliferate less, with more than 87% of cells in the G0 / G1 phase of the cell cycle, but are more sensitive to 4 different classes of drugs. In contrast, MCF7 cells in the high ATP subpopulation have significantly stronger proliferation capacity, with more than 38% of cells in the S phase or G2 / M phase, which indicates a significant multidrug resistance phenotype. Therefore, high levels of mitochondrial ATP are key drivers of cell proliferation and drug resistance because they represent the energetically "optimally survivable" cancer cell population.

[0129] The inventors have demonstrated that treatment with a panel of different anti-mitochondrial therapeutics i) metabolically induces ATP depletion and ii) is sufficient to effectively inhibit cancer cell metastasis using an in vivo xenograft animal model. These results suggest that high ATP levels are critical for the CSC metastatic process and are consistent with the data disclosed herein, indicating that high ATP CSCs are hyperproliferative, stem-like, anchorage-independent, and have increased antioxidant capacity and intrinsic multidrug resistance. Therefore, high ATP CSCs that can be isolated using this method may be responsible for tumor recurrence and metastasis in vivo.

[0130] The above bioinformatics analysis shows that ATP-related genes, especially ATP5F1C encoding the gamma subunit of the catalytic core of mitochondrial ATP synthase, are closely related to stemness, proliferation and metastasis. In addition, ATP5F1C is a prognostic biomarker for tumor recurrence and distal metastasis, and is also a marker for treatment failure of ER(+) patients treated with tamoxifen. In addition, high ATP MDA-MB-231 cells show a significant increase in the ability to carry out in vitro cell migration and invasion and spontaneous metastasis in vivo. Therefore, mitochondrial ATP plays a key role in metastatic dissemination. Therefore, mitochondrial ATP synthesis inhibitors should be effective for eradicating CSC in high ATP subpopulations as potential therapeutic agents for conveying metastasis prevention.

[0131] The pharmaceutical composition of the present method includes an ATP depletion compound (as identified above) in any pharmaceutically acceptable carrier. If a solution is required, water can be a selected carrier of a water-soluble compound or salt. Regarding water solubility, organic carriers such as glycerol, propylene glycol, polyethylene glycol or a mixture thereof may be suitable. In addition, without departing from the present method, a method for increasing water solubility can be used. In the latter case, the organic carrier can contain a large amount of water. The solution of either case can then be sterilized in a suitable manner known to those skilled in the art, and for illustration, filtered through a 0.22 micron filter. After sterilization, the solution can be distributed into a suitable container, such as a pyrogen-free glass vial. Distribution is optionally performed by an aseptic method. A sterilized closure can then be placed on the vial, and the vial contents can be lyophilized if necessary. Embodiments including second inhibitor compounds such as glycolysis inhibitors or OXPHOS inhibitors can co-administer the form of a second inhibitor available in the art. Unless otherwise indicated, it is not intended that the present method be limited to a specific form of administration.

[0132] In addition to the ATP depletion compound, the pharmaceutical preparation of the present method can also contain other additives known in the art. For example, some embodiments can include pH adjusting agents, such as acids (e.g., hydrochloric acid) and alkalis or buffers (e.g., sodium acetate, sodium borate, sodium citrate, sodium gluconate, sodium lactate, and sodium phosphate). Some embodiments can include antimicrobial preservatives, such as methylparaben, propylparaben, and benzyl alcohol. When the preparation is placed in a vial designed for multiple doses, an antimicrobial preservative is generally included. The pharmaceutical preparations described herein can be lyophilized using technology well known in the art.

[0133] In embodiments involving oral administration of ATP-depleting compounds, the pharmaceutical compositions may be in the form of capsules, tablets, pills, powders, solutions, suspensions, and the like. Tablets containing various excipients such as sodium citrate, calcium carbonate, and calcium phosphate may be used with various disintegrants such as starch (e.g., potato or tapioca starch) and certain complex silicates, and binders such as polyvinyl pyrrolidone, sucrose, gelatin, and gum arabic. In addition, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc may be included for tableting purposes. Solid compositions of similar types may be used as fillers for soft and hard-filled gelatin capsules. Materials in this regard also include lactose or milk sugar, and high molecular weight polyethylene glycols. When aqueous suspensions and / or elixirs are required for oral administration, the compounds of the presently disclosed subject matter may be combined with various sweeteners, flavoring agents, coloring agents, emulsifiers, and / or suspending agents, and diluents such as water, ethanol, propylene glycol, glycerol, and various combinations thereof. In embodiments having a carbocyanine compound and a second inhibitor compound, the second inhibitor compound may be administered in a separate form and is not limited to the form of the carbocyanine compound.

[0134] Additional embodiments provided herein include liposome preparations of ATP depletion compounds disclosed herein. The technology of forming liposome suspensions is well known in the art. When the compound is a water-soluble salt, conventional liposome technology is used, which can also be incorporated into lipid vesicles. In this case, due to the water solubility of the active compound, the active compound can be substantially trapped in the hydrophilic center or core of the liposome. The lipid layer used can have any conventional composition, and can contain cholesterol or can be cholesterol-free. When the active compound of interest is water-insoluble, conventional liposome formation technology is used again, and the salt can be substantially trapped in the hydrophobic lipid bilayer forming the liposome structure. In any case, the size of the liposome produced can be reduced, for example, by using standard ultrasound and homogenization techniques. The liposome preparation containing the active compound disclosed herein can be lyophilized to produce a lyophilized product, and the lyophilized product can be reconstituted with a pharmaceutically acceptable carrier such as water to regenerate the liposome suspension.

[0135] Regarding pharmaceutical compositions, the pharmaceutically effective amount of the ATP depletion compound herein should be determined by a health care practitioner, and should depend on the patient's health, body shape and age, and delivery route. In a non-limiting embodiment, a dosage of about 0.1 mg / kg to about 200 mg / kg has therapeutic efficacy, wherein the weight ratio is the weight of the ATP depletion compound (including the case of using salt) to the weight of the individual. In some embodiments, the dosage can be the amount of the compound required to provide a serum concentration of up to about 1 to 5, 10, 20, 30 or 40 μM of the active compound. In some embodiments, for oral administration, a dosage of about 1 mg / kg to about 10 mg / kg can be used, and in some embodiments, a dosage of about 10 mg / kg to about 50 mg / kg can be used. Typically, for intramuscular injection, a dosage of about 0.5 mg / kg to 5 mg / kg can be used. In some embodiments, for intravenous or oral administration, the dosage of the compound can be about 1 μmol / kg to about 50 μmol / kg, or optionally about 22 μmol / kg to about 33 μmol / kg. Oral dosage forms may include any suitable amount of active substance including, for example, 5 mg to 50, 100, 200 or 500 mg per tablet or other solid dosage form.

[0136] The following paragraphs describe the materials and methods used in conjunction with the data and embodiments described herein. It should be understood that alternative materials and methods generally accepted in the art may be used by those skilled in the art without departing from the present methods.

[0137] Cell lines and reagents: ER(+) [MCF7 and T47D] and triple-negative [MDA-MB-231 and MDA-MB-468] human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC). ATP-Red 1 (also known as BioTracker TM ATP-Red live cell dye; #SCT045) was purchased from Sigma-Aldrich, Inc.

[0138] Figure 1AThe heat map was prepared using the GSE36953 GEO dataset previously deposited in the NCBI database. Total RNA was prepared from MDA-MB-231 cells (TNBC cell line) under three different growth conditions: 2D adherent growth, 3D anchorage-independent growth, and in vivo tumor growth. Affymetrix Human Genome U133 Plus 2.0 arrays were used for analysis. The heat map was generated using QIAGEN OmicSoft Suite software. ATP-related genes were transcriptionally upregulated relative to 2D adherent growth under both 3D growth conditions (anchorage-independent and in vivo tumors).

[0139] Flow cytometry after Vital Staining with ATP-Red 1: Human breast cancer cell lines were first grown as 2D monolayers or 3D spheroids. The cells were then collected and dissociated into single-cell suspensions and then analyzed or sorted by flow cytometry using a SONYSH800 cell sorter. In short, after vital staining with the probe ATP-Red 1, high ATP cell subpopulations and low ATP cell subpopulations were separated. Within the ATP-Red 1 signal, high ATP cell subpopulations and low ATP cell subpopulations were selected by gating. Unless otherwise stated, cells with the lowest (last 5% or 10%) fluorescence signal or the highest (first 5% or 10%) fluorescence signal were collected as low ATP and high ATP, respectively. Due to the gating setting, cells outside the gate were discarded during the sorting process. However, such a setting is usually required to ensure high purity during the sorting process. Data were analyzed using FlowJo 10.1 software.

[0140] ATP Assay with Cell-Titer-Glo: Cell-Titer-Glo (#G7570) was obtained from Promega, Inc. and used to measure ATP levels in lysed cells according to the manufacturer's recommendations. Cell-Titer-Glo is a luciferase-based assay system.

[0141] 3D anchorage-dependent growth assay: Single cell suspensions were prepared using enzymatic (1x Trypsin-EDTA, Sigma Aldrich, catalog number T3924) and manual disaggregation (25-gauge needle). Under non-adherent conditions, 5000 cells were plated in mammosphere medium (DMEM-F12 / B27 / 20ng / ml EGF / PenStrep) in six-well plates coated with 2-hydroxyethyl methacrylate (poly-HEMA, Sigma, catalog number P3932). Cells were grown for 5 days and maintained in a humidified incubator at 37°C in 5% (v / v) carbon dioxide / air at atmospheric pressure. After 5 days, 3D spheres with a diameter greater than 50 μm were counted using a microscope equipped with a graduated eyepiece, and the percentage of cells that formed spheres was calculated and normalized to 1 (1 = 100% MFE; mammosphere formation efficiency). The mammosphere assay was performed in triplicate and repeated three times.

[0142] Metabolic flux analysis: Extracellular acidification rate and oxygen consumption rate were analyzed using a Seahorse XFe96 analyzer (Agilent / Seahorse Bioscience, USA). Cells were maintained in DMEM supplemented with 10% FBS (fetal bovine serum), 2mM GlutaMAX, and 1% Pen-Strep. 20,000 breast cancer cells were seeded into each well of an XFe96-well cell culture plate and incubated at 37°C in a 5% CO2 humidified atmosphere for at least 12 hours to allow the cells to attach. After approximately 24 hours, MCF7 cells were washed in preheated XF assay medium or used for OCR measurement, and the XF assay medium was supplemented with 10mM glucose, 1mM pyruvate, 2mM L-glutamine and adjusted to 7.4 pH. The cells were then kept in 175 μL / well XF assay medium in a non-CO2 incubator at 37°C for 1 hour. During the incubation period, 25 μL 80mM glucose, 9 μM oligomycin and 1M 2-deoxyglucose (for ECAR measurement) or 10 μM oligomycin, 9 μM FCCP, 10 μM rotenone, 10 μM antimycin A (for OCR measurement) were loaded into the injection port of the XFe96 sensor box in XF assay medium. The measurement results were normalized by protein content (SRB assay) and Hoechst33342 content. Using XFe96 software and GraphPad Prism software, single-factor ANOVA and student t-test were used to calculate and analyze data sets. All experiments were performed three times independently in quintuplicate.

[0143] Cell cycle analysis by FACS: Cell cycle analysis of high and low ATP cell subpopulations was performed by FACS analysis using an Attune NxT flow cytometer. Briefly, after trypsinization, resuspended cells were incubated with 10 ng / ml Hoescht solution (Thermo Fisher Scientific) at 37°C in the dark for 40 min. After 40 minutes, cells were washed and resuspended in PBS Ca / Mg for acquisition or resuspended in sorting buffer [1× PBS containing 3% (v / v) FBS and 2 mM EDTA] for FACS. 50,000 events were analyzed for each condition. Gated cells were manually sorted into multiple cell cycle phases.

[0144] Statistical significance: All analyses were performed using GraphPad Prism 6. Data are expressed as mean ± standard deviation (or ± SEM, where indicated). All experiments were performed at least 3 times independently, with >3 technical replicates for each experimental condition tested (unless otherwise stated, e.g., when representative data are shown). Statistically significant differences were determined using Student's t-test or analysis of variance (ANOVA). For comparisons between multiple groups, one-way ANOVA was used to determine statistical significance. p<0.05 was considered significant, and all statistical tests were two-way: p*<0.05; p**<0.01; p***<0.005; p****<0.0001.

[0145] Bioinformatics analysis: Unbiased label-free proteomics comparing 2D monolayers and 3D mammospheres was performed using MCF7 and T47D breast cancer cell lines as described previously. Informatics analysis was performed using various publicly available GEO datasets (GSE36953, GSE2034, GSE59000, GSE55470) archived in the NCBI database related to 3D growth, metastasis, and circulating tumor cells (CTCs). Gene expression profiling data were extracted from these GEO datasets. Heatmaps were generated using QIAGEN OmicSoft Suite software. Volcano plots were generated by examining the annotations present in Oncology and Metastatic Cancer (QIAGEN OmicSoft Suite). In addition, functional “core analysis” of annotated genes was performed using Ingenuity Pathway Analysis software (IPA; QIAGEN). Gene co-expression profiles were extracted from The Metastatic Breast Cancer Project Provisional (2020) using cBioPortal (https: / / www.cbioportal.org / ); mRNA expression profiling (RNA Seq V2 RSEM) was performed by RNA sequencing of metastatic breast cancer samples from 146 patients.

[0146] Kaplan-Meier (KM) analysis: To perform a KM analysis of ATP5F1C, we used an open access online survival analysis tool to interrogate publicly available microarray data from up to 3,951 breast cancer patients. To this end, we primarily analyzed data from ER(+) patients. Biased array data were excluded from the analysis. This allowed us to identify ATP5F1C (also known as ATP5C1) as a meaningful prognostic marker. Hazard ratios were calculated at the best automatically selected cutoff values, and p-values ​​were calculated using the log-rank test and plotted in R. KM curves were generated online (as high-resolution TIFF files) using the KM plotter using univariate analysis:

[0147] https: / / kmplot.com / analysis / index.php?p=service&cancer=breast .

[0148] This approach allowed direct in silico validation of ATP5F1C as a marker of tumor recurrence (RFS, recurrence-free survival) and distant metastasis (DMFS, distant metastasis-free survival). All these analyses used the latest 2020 version of the database.

[0149] Cell migration assay: Briefly, 2.5 × 10 4Cells were added to the wells of 8-μm pore, non-coated membrane-modified Boyden chambers (Transwells). The lower chamber contained 10% fetal bovine serum in DMEM as a chemical attractant. The cells were incubated at 37°C and allowed to migrate for a full 6-hour period. Non-invasive cells were removed from the upper surface of the membrane by wiping with a cotton swab. The chamber was stained in 0.5% crystal violet diluted in 100% methanol for 30-60 min, rinsed in water and examined under a bright field microscope. The values ​​of invasion and migration were obtained by calculating five fields of view (20× objective) for each membrane and represent the average of three independent experiments. Please note that Transwells pre-coated with extracellular matrix (i.e., Matrigel) were used to measure invasive cell invasion and prevent simple cell migration.

[0150] Transfer assay: Chicken embryo transfer assay was performed by INOVOTION (Société: 811310127), La Tronche-France. According to French legislation (decree n°2013–118, 1 February 2013; art. R-214–88), no ethical approval is required for scientific experiments using oviparous embryos. Animal studies were performed under animal experimentation permits N°381029 and B3851610001 of INOVOTION. Fertilized White Leghorn eggs were incubated at 37.5°C and 50% relative humidity for 9 days. More than 20 eggs were processed for each experimental condition. At that moment (E9), the chorioallantoic membrane (CAM) was lowered by drilling a small hole in the eggshell to access the air sac and a 1 cm 2 window. The MDA-MB-231 tumor cell line was cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin. On day E9, the cells were detached with trypsin, washed with complete medium, and suspended in transplantation medium. After ATP-based cell sorting by flow cytometry, an inoculum of 30,000 cells was added to the CAM of each egg (E9), and the eggs were then randomly grouped. On day E18, a 1 cm2 section of the lower CAM was collected to assess the number of metastatic cells in 8 samples per group (n=10). Genomic DNA in the CAM was extracted (commercial kit) and analyzed by qPCR using specific primers for human Alu sequences. By CFX Maestro software directly handles the Cq calculation, average Cq and relative transfer for each sample in each group. Uninjected eggs were also evaluated in parallel as a specific negative control. All data were subjected to one-way ANOVA and post hoc tests.

[0151] The terms used in describing the embodiments of the present method are only used for the purpose of describing specific embodiments and are not intended to be limiting. As used in the specification and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. As will become apparent by considering the following detailed description, the present method includes many alternatives, modifications, and equivalents.

[0152] It should be understood that although the terms "first", "second", "third", "a)", "b)" and "c)" etc. can be used in this article to describe various elements of the present method, the claims should not be limited by these terms. These terms are only used to distinguish one element of the present method from another element. Therefore, without departing from the teachings of the present method, the first element discussed below can be referred to as an element aspect, and similarly, the third element can also be referred to as an element aspect. Therefore, the terms "first", "second", "third", "a)", "b)" and "c)" etc. are not intended to necessarily convey the order or other hierarchy of related elements, but are only used for identification purposes. The order of operations (or steps) is not limited to the order presented in the claims.

[0153] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as those commonly understood by those of ordinary skill in the art. It should also be understood that terms such as those defined in common dictionaries should be interpreted as having the same meaning as in the context of the present application and the related art, and should not be interpreted in an idealized or overly formal sense, unless so clearly defined herein. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict in terms, this specification shall prevail.

[0154] Furthermore, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as no combinations when interpreted in the alternative ("or").

[0155] Unless the context indicates otherwise, it is specifically intended that the various features of the present method described herein can be used in any combination. In addition, the present method also contemplates that in some embodiments, any feature or feature combination described for the illustrative embodiments can be excluded or omitted.

[0156] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be interpreted as including the recited materials or steps "and those that do not materially affect the basic and novel characteristics of the claim." Therefore, the term "consisting essentially of" as used herein should not be interpreted as being equivalent to "comprising."

[0157] The term "about" as used herein refers to a measurable value, such as an amount or concentration, etc., and is intended to include variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount. The ranges of measurable values ​​provided herein may include any other ranges and / or individual values ​​therein.

[0158] Having thus described certain embodiments of the present method, it should be understood that the scope of the appended claims is not to be limited to the specific details set forth in the foregoing description, since many obvious variations thereof are possible without departing from the spirit or scope thereof as hereinafter claimed.

Claims

1. A purified cell composition, the composition consisting primarily of a subpopulation of cancer stem cells stained with a fluorescent adenosine triphosphate (ATP) imaging probe and expressing a target portion of a cancer cell population's ATP-based fluorescence signal range, the subpopulation being separated from the cancer cell population, wherein the target portion is: (a) one of the top 10% of the ATP-based fluorescence signal or the top 5% of the ATP-based fluorescence signal; and / or (b) one of the bottom 10% of the ATP-based fluorescence signal or the bottom 5% of the ATP-based fluorescence signal.

2. The composition of claim 1, wherein the cancer cell population is a human cancer cell population that expresses a range of fluorescent signals in response to a fluorescent ATP imaging probe, and the cell subpopulation expresses an upper portion of the ATP-based fluorescent signal range, wherein the cancer stem cell subpopulation is isolated from the human cancer cell population and comprises hyperproliferative cancer stem cells, and wherein the upper portion comprises: (a) the top 10% of the fluorescence signal based on ATP; or (b) Top 5% of ATP-based fluorescence signal.

3. The composition according to claim 2, wherein: (a) the cell subpopulation is stained with ATP-Red 1; and / or (b) The composition is frozen.

4. A composition according to any preceding claim, wherein: (a) the composition is positive for one of a CD44 marker or an ALDH marker; and / or (b) The cell subpopulations were stained with fluorescent ATP imaging dye.

5. The composition according to any one of claims 1-3, wherein the cancer stem cell subpopulation is obtained by staining a human cancer cell population with a fluorescent ATP imaging probe, isolating a portion of the human cancer cell population having a target portion of an ATP-based fluorescent signal, and purifying the isolated cells.

6. The composition of claim 4, wherein the cancer stem cell subpopulation is obtained by staining a human cancer cell population with a fluorescent ATP imaging probe, isolating a portion of the human cancer cell population having a target portion of an ATP-based fluorescent signal, and purifying the isolated cells.

7. The composition according to claim 5, wherein: (a) the target portion comprises one of the top 10% of ATP-based fluorescent signals or the top 5% of ATP-based fluorescent signals, and the isolated cells are positive for one of a CD44 marker or an ALDH marker; and / or (b) The fluorescent ATP imaging probe includes ATP-Red 1.

8. The composition according to claim 6, wherein: (a) the target portion comprises one of the top 10% of ATP-based fluorescent signals or the top 5% of ATP-based fluorescent signals, and the isolated cells are positive for one of a CD44 marker or an ALDH marker; and / or (b) The fluorescent ATP imaging probe includes ATP-Red 1.

9. A cell fractionation method based on ATP, the method comprising: staining cells in the cancer cell population with a fluorescent ATP imaging probe that fluoresces when bound to ATP; measuring an ATP-based fluorescence signal of stained cells in the cancer cell population; and separating the stained cells according to the target portion of the ATP-based fluorescent signal, The target portion includes the top 10% of the fluorescent signal based on ATP or one of the top 5% of the fluorescent signal based on ATP and / or the bottom 10% of the fluorescent signal based on ATP or one of the bottom 5% of the fluorescent signal based on ATP.

10. The method of claim 9, wherein separating the stained cells according to the target portion of the ATP-based fluorescent signal comprises performing fluorescence activated cell sorting (FACS) gating on the target portion of the ATP-based fluorescent signal.

11. The method of claim 10, wherein the gate is set to collect at least one of: (i) stained cells having the top 10% of the measured ATP-based fluorescent signal and (ii) stained cells having the bottom 10% of the measured ATP-based fluorescent signal.

12. The method of claim 9, wherein the fluorescent ATP imaging probe comprises ATP-Red 1.

13. The method of claim 9, wherein the cell population is derived from one of blood, urine, saliva, tumor tissue, non-cancerous tissue, and metastatic lesions.

14. The method of claim 9, further comprising at least one of: measuring ALDH activity of the isolated cells, measuring anchorage-independent growth of the isolated cells, measuring mitochondrial mass of the isolated cells, measuring glycolytic metabolism and oxidative mitochondrial metabolism of the isolated cells, measuring cell cycle progression and proliferation rate of the isolated cells, and measuring polyploidy of the isolated cells.

15. The method of any one of claims 9-14, wherein the method comprises isolating and collecting metabolically hyperproliferative cells from the cell population, and wherein the ATP imaging probe is an ATP-labeling dye, the method further comprising collecting at least a portion of the isolated cells having a measured ATP-based fluorescent signal that is one of above a predetermined threshold and below a predetermined threshold. The method of claim 15 , wherein the ATP labeling dye comprises ATP-Red 1.

17. The method of claim 15, wherein the predetermined threshold comprises a portion of an upper portion of the measured ATP-based fluorescent signal.

18. The method of claim 17, wherein the predetermined threshold comprises one of top 10%, top 5%, top 2%, and top 1%.

19. The method of claim 15, wherein separation and collection are performed using fluorescence activated cell sorting (FACS).

20. The method of claim 15, wherein the isolated cells are further separated based on a second marker.

21. The method of claim 20, wherein the second marker comprises one of the following: CD44(+), CD133(+), ESA(+), ALDEFLOUR(+), MitoTracker-High, EpCAM(+), CD90(+), CD34(+), CD29(+), CD73(+), CD90(+), CD105(+), CD106(+), CD166(+), and Stro-1(+).

22. The method of claim 21, wherein the cell separation based on the second marker occurs in at least one of the following situations: (i) before staining the cells in the cell population with the ATP labeling dye, and (ii) after staining the cells in the cell population with the ATP labeling dye.

23. The method of claim 22, wherein the second marker comprises an antibody coated on magnetic beads.

24. The method of claim 15, further comprising staining cells in the cell population with a second marker, and wherein the measuring of the ATP-based fluorescent signal of the stained cells in the cell population occurs after staining with the second marker and the ATP labeling dye.

25. Use of the purified cell composition according to any one of claims 1 to 8 in preparing a kit for testing the anti-cancer activity of a candidate compound.

26. A cell composition obtained by the method of any one of claims 9-24.

27. A method for testing the anti-cancer activity of a candidate compound, the method comprising: staining the cancer cell population with an ATP-labeling dye that fluoresces when bound to ATP; measuring the ATP-based fluorescence signal of the stained cells; separating the stained cells according to a target portion of the ATP-based fluorescent signal to prepare a hyperactive cancer cell subpopulation, wherein the target portion is one of the top 10% of the ATP-based fluorescent signal or the top 5% of the ATP-based fluorescent signal; administering the candidate compound to the hyperactive cancer cell subpopulation; and The effect of the candidate compound on the hyperactive cancer cell subpopulation is measured.

28. The method of claim 27, wherein the ATP labeling dye comprises ATP-Red 1.

29. The method of claim 27, wherein the target portion of the ATP-based fluorescent signal comprises one of the top 10%, the top 5%, the top 2%, and the top 1%.

30. The method of claim 27, wherein the hyperactive cancer cell subpopulation is positive for one of a CD44 marker and an ALDH marker.

31. The method of claim 27, wherein the method further comprises at least one of: measuring ALDH activity of the hyperactive cancer cell subpopulation, measuring anchorage-independent growth of cells of the hyperactive cancer cell subpopulation, measuring mitochondrial mass of the hyperactive cancer cell subpopulation, measuring glycolytic metabolism and oxidative mitochondrial metabolism of the hyperactive cancer cell subpopulation, measuring cell cycle progression and proliferation rate of the hyperactive cancer cell subpopulation, and measuring polyploidy of the hyperactive cancer cell subpopulation.

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