WO2011109651A2 - Expression vectors for classifying cells as cell cycling or hypoxic and methods of use - Google Patents

Abstract

The invention provides compositions for detectably identifying cells as actively dividing and/or as hypoxic, and provides methods of using these compositions for the classification and isolation of said cells. Such compositions are further useful in methods of drug screening.

Description

EXPRESSION VECTORS FOR CLASSIFYING CELLS AS CELL CYCLING

OR HYPOXIC AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of the following U.S. Provisional Application No.:

61/310,138, filed March 3, 2010, the entire contents of which are incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

This work was supported by the following grant from the National Institutes of Health, Grant No: 5R01CA057341-20. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Over the past decade, links between cancer genes and altered cancer metabolism are emerging with profound therapeutic implications. Despite the emerging interest in this area, much remains to be learned about cancer cell genomics and metabolism in the context of the tumor environment. This is largely due to the limitation in being able to isolate

subpopulations of cancer cells that have specific characteristics to study their genomic responses and proteomes as clues to their susceptibility to cancer treatment. In particular, the tumor microenvironment is heterogeneous, comprising cycling and non-cycling cells that could be hypoxic or non-hypoxic. The ability to isolate defined subpopulations of cancer cells remains an obstacle to on-going cancer research and the development of therapeutics. SUMMARY OF THE INVENTION

As described below, the present invention features compositions and methods featuring combinations of fluorescent proteins that identify cells that are hypoxic as well, as those that are cycling. The coupling of these two fluorescent beacons provides methods for characterizing the tumor microenvironment, classifying subpopulations of cancer cell having differing metabolisms, and characterizing the chemotherapeutic responses of subpopulations of cancer cells.

In one aspect, the invention provides an isolated nucleic acid molecule having a bidirectional reporter cassette that encodes two reporters including a first reporter polypeptide fused to a destabilizing moiety, where the first reporter selectively identifies a cycling cell and a second reporter polypeptide fused to a destabilizing moiety, where the second reporter selectively identifies a hypoxic cell, where the expression of the second reporter polypeptide is under the control of a regulatory element that is selectively expressed under hypoxic conditions.

In various embodiments of any of the aspects as described herein, each of the two reporters is detectable by fluorescence. In various embodiments of any of the aspects described herein, the where the fluorescence emitted by the reporters is at distinct and distinguishable wave lengths. In various embodiments of any of the aspects described herein, the isolated the first and second reporter are selected from the group consisting of GFP, RFP, BFP, CFP, YFP, mCherry, and EvoGlow. In various embodiments of any of the aspects described herein, the first reporter is GFP and the second reporter is mCherry.

In various embodiments of any of the aspects described herein, the destabilizing moiety is selected from the group consisting of PEST domain, geminin motif, or fragments or analogs thereof. In various embodiments of any of the aspects described herein, the the regulatory element is a hypoxic responsive element, an oxygen dependent domain (ODD) of HIF-Ι , or fragments or analogs thereof. In various embodiments of any of the aspects described herein, the ODD is HIF- la residues 548-603, 530-603, or 400-620. In various embodiments of any of the aspects described herein, the first and second reporter

polypeptides have a half-life that is about equal to the time required for cell division. In various embodiments of any of the aspects described herein, the first reporter polypeptide has a half life that is about 5, 10, 15, 20, 24, or 26 hours. In various embodiments of any of the aspects described herein, the the second reporter polypeptide has a half life that is about 3, 4, 5, 6, 7, 8, 9, or 10 hours.

In various embodiments of any of the aspects described herein, the first reporter polypeptide is under the control of a promoter that is selectively expressed in dividing cells. In various embodiments, the promoter is the CMV promoter, beta-actin promoter, SV40 promoter-enhancer, or phosphoglycerate kinase (PGK) promoter.

In various embodiments of any of the aspects described herein, the first reporter fused to the destabilizing moiety is separated from the regulatory elements directing expression of the second reporter by an insulator or insulating polynucleotide sequence. In various embodiments of any of the aspects described herein, the promoter controlling expression of the first reporter and the regulatory element controlling expression of the second reporter are separated from surrounding nucleic acid sequences by an insulating polynucleotide sequence. In various embodiments, the insulator is between about 100 and 2000 nucleic acids in length (e.g., 100, 200, 250, 500, 750, 1000, 1250, 1500, 1750, 2000) or even longer. In particular embodiments, the insulating polynucleotide sequence is about 1500 kb in length.

In various embodiments of any of the aspects as described herein, each of the two bidirectional reporters is linked to a selectable marker. In various embodiments of any of the aspects described herein, the selectable marker is puromycin, hygromycin, or neomycin.

In one aspect, the invention provides a vector comprising an isolated nucleic acid molecule having a bidirectional reporter cassette of any of the aspects as described herein. In various embodiments of any of the aspects described herein, the vector is an expression vector suitable for expression in a mammalian cell. In various embodiments of any of the aspects described herein, the expression vector is a viral or non-viral expression vector. In various embodiments of any of the aspects described herein, the viral expression vector is derived from a lentivirus, adenovirus, retrovirus, adeno-associated virus, herpesvirus, vaccinia virus or polyoma virus.

In an additional aspect, the invention provides an expression vector having a bidirectional reporter cassette that encodes a first reporter having a HIF-1 oxygen dependent domain (ODD) fused to a GFP polypeptide fused to a PEST moiety and a second reporter having an mCherry polypeptide fused to a geminin polypeptide, where the expression of the second reporter polypeptide is under the control of an HRE regulatory element and where an insulator sequence is located between the two reporters.

In another aspect, the invention provides an expression vector having a bidirectional reporter cassette that encodes a first reporter having a HIF-1 oxygen dependent domain (ODD) fused to an evoglow polypeptide fused to a PEST moiety and a second reporter having an mCherry polypeptide fused to a geminin polypeptide, where the expression of the second reporter polypeptide is under the control of an HRE regulatory element and where an insulator sequence is located between the two reporters.

In an additional aspect, the invention provides an expression vector having a bidirectional reporter cassette that encodes a first reporter having a HIF-1 oxygen dependent domain (ODD) fused to a GFP polypeptide fused to a PEST moiety and a second reporter having an mCherry polypeptide fused to a geminin polypeptide, where the expression of the second reporter polypeptide is under the control of an HRE regulatory element, where an insulator sequence is located between the two reporters, and where the bidirectional reporter cassette is located between two insulator sequences.

In yet another aspect, the invention provides an expression vector having a bidirectional reporter cassette that encodes a first reporter having a HIF-1 oxygen dependent domain (ODD) fused to an evoglow polypeptide fused to a PEST moiety and a second reporter having an mCherry polypeptide fused to a geminin polypeptide, where the expression of the second reporter polypeptide is under the control of an HRE regulatory element and where an insulator sequence is located between the two reporters, and where the bidirectional reporter cassette is located between two insulator sequences.

In still another aspect, the invention provides an expression vector having a reporter cassette that encodes a first reporter having a HIF-1 oxygen dependent domain (ODD) fused to a GFP polypeptide fused to a PEST moiety and a second reporter having an mCherry polypeptide fused to a geminin polypeptide, where the expression of the second reporter polypeptide is under the control of an HRE regulatory element, where the first reporter is located upstream of the second reporter, and where an insulator sequence is located upstream of the first reporter.

In another aspect, the invention provides a cell or host cell containing a vector comprising an isolated nucleic acid molecule having a bidirectional reporter cassette of any of the aspects as described herein. In various embodiments of any of the aspects described herein, the cell is in vitro, in vivo, or ex vivo. In various embodiments of any of the aspects described herein, the cell is a mammalian cell. In various embodiments of any of the aspects described herein, the cell is a human cell. In various embodiments of any of the aspects as described herein,the cell is derived from a tumor or immortalized cell line. In various embodiments of any of the aspects described herein, the cell is a HeLa cell, 293T cell, P493 lymphoma cell, or a PI 98 cell.

In another aspect, the invention provides a xenograft containing a cell having a vector of any of the aspects as described herein.

In another aspect, the invention provides a transgenic non-human animal having an expression vector containing a bidirectional reporter cassette that encodes a first reporter polypeptide fused to a destabilizing moiety, where the first reporter selectively identifies a cycling cell and a second reporter polypeptide fused to a destabilizing moiety, where the second reporter selectively identifies a hypoxic cell, where the expression of the second reporter polypeptide is under the control of a regulatory element that is selectively expressed under hypoxic conditions. In various embodiments of any of the aspects described herein, the animal is a mammal. In various embodiments of any of the aspects described herein, the mammal is a rodent. In various embodiments of any of the aspects described herein, the rodent is a mouse or rat. In another aspect, the invention provides a method for classifying tumor cells as hypoxic cells, cycling cells, or cycling hypoxic cells, the method involving expressing in the cells an expression vector of any of the aspects as described herein; and detecting the expression of the first and second reporters in the cells. In various embodiments of any of the aspects described herein, the method further comprises characterizing the expression of a polypeptide selected from the group consisting of HIF, MYC, HK2, PKM2, LDHA, PDK1, MCT1, GLUD1 (glutamate dehydrogenase), and GPT (glutamate pyruvate transaminase) or the polynucleotides encoding them.

In another aspect, the invention provides a method for isolating one or more tumor cell subpopulations, each cell subpopulation comprising hypoxic, cycling, or cycling hypoxic cells, the method involving expressing in the cells an expression vector of any of the aspects as described herein; detecting the expression of the first and second reporters in the cells; and isolating a population of cells enriched for expression of the first reporter, isolating a subpopulation of cells enriched for expression of the second reporter, and/or isolating a population of cells enriched for expression of the first and second reporters, where each of the cell subpopulations are enriched for hypoxic, cycling, or cycling hypoxic cells, respectively. In various embodiments of any of the aspects described herein, the cells are isolated using fluorescence activated cell sorting (FACS) or Laser-Enabled Analysis and Processing (LEAP) microplate -based cytometry. In various embodiments of any of the aspects described herein, the first and second reporters are fluorescent proteins that emit at distinct and distinguishable wave lengths.

In another aspect, the invention provides a method for characterizing the

chemotherapuetic activity of an agent, the method involving expressing in a population of cells an expression vector of any of the aspects as described herein, where the population comprises cells exposed to normoxic and hypoxic conditions; contacting the cells with a chemotherapeutic agent; detecting an alteration in the survival or the proliferation of the cells; detecting the expression of the first and second reporters in any surviving cells, where the disproportionate survival of a cell expressing a hypoxic reporter characterizes the chemotherapeutic agent as ineffective in reducing the survival or proliferation of hypoxic cells, and the disproportionate survival of cycling cells characterizes the chemotherapeutic agent as ineffective in reducing the survival or proliferation of cycling cells.

In another aspect, the invention provides a method for identifying an agent that reduces the proliferation or survival of a hypoxic cell that is refractory to conventional chemotherapy, the method involving expressing in a population of cells an expression vector of any of the aspects as described herein, where the population comprises cells exposed to normoxic and hypoxic conditions; contacting the cells with a chemotherapeutic agent and detecting a reduction in the survival or the proliferation of the cells; detecting the expression of a hypoxic reporter in the surviving cells, and exposing the surviving cells of step (c) to a second agent, and detecting a reduction in the survival or proliferation of the surviving cells of step (c), thereby identifying the agent as reducing the proliferation or survival of a hypoxic cell that is refractory to conventional chemotherapy. In various embodiments of any of the aspects described herein, the chemotherapeutic agent is hydroxyurea or another agent that inhibits ribonucleotide reductase or otherwise inhibits cell cycling. In various embodiments of any of the aspects as described herein, the chemotherapeutic agent is gemcitabine. In various embodiments of any of the aspects as described herein, the second agent is Cytoxan or an LDHA inhibitor. In various embodiments of any of the aspects described herein, the cell is derived from a tumor or immortalized cell line. In various embodiments of any of the aspects described herein, the cell is a HeLa cell, 293T cell, P493 lymphoma cell, or a PI 98 cell.

The invention provides compositions and methods that provide for the identification and characterization of tumor cells using a combination of fluorescent proteins that identify cells that are hypoxic as well as those that are cycling. Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

The present application is related to U.S. Patent Application Serial Nos. 11/664,883, 11/921,532, and 13/002,202; International Patent Application Nos. PCT/US05/36067, PCT/US09/03930, and PCT/US06/21613; and U.S. Provisional Patent Application Serial Nos. 60/617,610, 60/698,795, 60/687,488, 60/687,756, and 61/142,985. The aforementioned applications are incorporated herein by reference in their entirety.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide (e.g., reporter polypeptide) as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical

modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

As used herein, "bidirectional" is meant to refer to the orientation of two sequences whose transcription occurs in opposite directions. As used herein, "unidirectional" is meant to refer to the orientation of two reporters in a reporter cassette such that their transcription occurs in the same direction. For example, a first reporter is positioned downstream of a second reporter such that both reporters are transcribed in the same direction. As used herein, "downstream" is meant to refer to the positioning of a first nucleic acid sequence 3' to a second nucleic acid sequence with reference to the direction of transcription of the second nucleic acid sequence. As used herein, "upstream" is meant to refer to the positioning of a first nucleic acid sequence 5' to a second nucleic acid sequence with reference to the direction of transcription of the second nucleic acid sequence.

As used herein, "cassette" or "reporter cassette" means a DNA sequence capable of directing expression of a nucleotide sequence in a cell. In one embodiment, a cassette comprises a promoter operably linked to a nucleotide sequence of interest that is optionally operably linked to termination signals and/or other regulatory elements. A cassette may also comprise sequences required for proper translation of the nucleotide sequence. In certain embodiments of the invention a cassette comprises nucleic acid sequences encoding two reporters that are expressed. In particular embodiments, expression of a first reporter selectively identifies a cycling cell and expression of a second reporter identifies a hypoxic cell. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. For example, the detectable reporter polypeptide may be operably linked to one or more of a degradation moiety (e.g., PEST), cell-cycle polypeptide (e.g., geminin), or hypoxia responsive polypeptide (e.g., HIF-1 oxygen dependent domain) to form a fusion polypeptide. An expression cassette may be assembled entirely extracellularly (e.g., by recombinant cloning techniques). The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter which initiates transcription only when the host cell is exposed to some particular stimulus (e.g., a promoter comprising hypoxic responsive elements). In the case of a multicellular organism, expression of a reporter in the cassette can be specific to a particular microenvironment, tissue, organ, or stage of development.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes,"

"including," and the like; "consisting essentially of" or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

"Detect" refers to identifying the presence, absence or amount of the analyte to be detected.

By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include any neoplasia known in the art.

As used herein, "destabilizing moiety" is meant a moiety that reduces the half-life of the polypeptide comprising the moiety. In one embodiment, the destabilizing moiety promotes the degradation of the protein. Preferably the half-life of a polypeptide comprising a destabilizing moiety is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In one embodiment, the the half-life of a polypeptide comprising a destabilizing moiety is about is about equal to the time required for cell division or 5, 10, 15, 20, 24, or 26 hours.

By "enhancer", as used herein, refers to a regulatory nucleic acid sequence, which can function in either orientation and in any location with respect to a promoter, to modulate (e.g., increase) the effect of a promoter (e.g., to increase transcription levels). For example, an enhancer of the present invention may act in a position-independent and an

orientationindependent manner to induce transcription of an operatively linked

polynucleotide.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. As used herein, "hypoxia" or "hypoxic" is meant a lower level of oxygen or oxygen concentration in a cell or tissue compared to what is normally found (e.g., below normoxic or physiological levels). For example, conventional culture systems typically use 20% oxygen. In vivo cells typically have 6% 0₂ . Hypoxic levels are below about 6%, 5%, 2%, 1%, 0.5% concentration 0₂, or 0% concentration 0₂. Hypoxia relates to an oxygen deficiency in bodily tissues. Cells or tissues are hypoxic when the 0₂ concentration is lower than the normal level of oxygen in these particular cells or tissues. In relation to in vitro experiments, "hypoxia" means below physiological levels.

By "insulator" or "insulating sequence" is meant a nucleic acid sequence that decreases or prevents the influence of other nearby DNA sequences on transcription.

Insulators, for example, prevent crosstalk of promoter or enhancer sequences in close proximity to each other. Insulator sequences have been described, for example, in Gaszner and Felsenfeld, "Insulators: exploiting transcriptional and epigenetic mechanisms." Nat Rev Genet. 2006 Sep;7(9):703-13 and Burgess-Beusse, B, et al. "The insulation of genes from external enhancers and silencing chromatin". Proc. Natl Acad. Sci. USA 9 (Suppl 4): 16433- 16437.

By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

By "neoplasia" is meant a disease or disorder characterized by excess proliferation or reduced apoptosis. Illustrative neoplasms for which the invention can be used include, but are not limited to leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblasts leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangio sarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma,

choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and

retinoblastoma).

As used herein, "normoxia" or "normoxic" is meant a normal or physiological level of oxygen supply to bodily tissues. In one embodiment, a normoxic concentration is 6% 0₂.

As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

By "promoter" is meant a polynucleotide sufficient to direct transcription.

By "operably linked" is meant that a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide.

By "reporter" is meant a molecule (e.g., a polypeptide) that is detectable or has a detectable property (e.g., fluorescence). In the reporter cassettes of the invention, the coding region encodes a reporter polypeptide. A "detectable reporter" is a polypeptide that comprises a moiety that renders it detectable, via any means, including spectroscopic, photochemical (e.g., lucif erase, GFP), biochemical, immunochemical, or chemical means. Detectable reporter polypeptides of the invention include for example GFP , evoglow, mCherry, and RFP.

By "regulatory element" or "regulatory sequence" is meant a nucleic acid which, when operably linked to a polynucleotide, modulates transcription and/or expression levels of the polynucleotide in a cell. Genetic regulatory elements of the present invention may include promoters, enhancers, insulators, or a combination thereof, as well as other cis-acting sequences involved in the binding of transcription factors. Regulatory elements include both positive and negative regulators of transcription.

As used herein, the terms "selectable marker" or "selectable marker gene" is meant a nucleic acid sequence that confers a particular phenotype upon a cell. In one embodiment, the selectable marker confers resistance to an antibiotic or drug. In another embodiment, the selectable marker provides an enzymatic activity that confers the ability to grow in medium lacking a nutrient. Antibiotic selectable markers used in the vectors of the invention include resistance genes for puromycin, hygromycin, or neomycin. When a host cell must express a selectable marker to grow in selective medium, the marker is said to be a positive selectable marker (e.g., antibiotic resistance genes which confer the ability to grow in the presence of the appropriate antibiotic). Selectable markers can also be used to select against host cells containing a particular gene; selectable markers used in this manner are referred to as negative selectable markers.

By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

By "reference" is meant a standard or control condition.

A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule.

By "hybridize" is meant pair to form a double-stranded molecule between

complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.

Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;

aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e^"3 and e^"100 indicating a closely related sequence.

By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, feline, or rodent.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural. Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C depict how microenvironment can cause tumor heterogeneity. Figure 1A is a diagram depicting glucose and glutamine metabolic pathways and targets (boxed) therein regulated by Myc (dashed arrows). Glucose is transported by Glutl and

phosphorylated by hexokinase 2 (HK2) with subsequent conversion to trioses, producing NADH and ATP, culminating in pyruvate. Intermediate trioses yield glycerol-3-phosphate as a backbone for lipids. Pyruvate can be converted to lactate by lactate dehydrogenase A (LDHA), which is a target of Myc and HIF-1. In the presence of oxygen, pyruvate could be further converted to acetyl-CoA (AcCoA) that is further oxidized in the mitochondria through the tricarboxylic acid (TCA) cycle, which donates high energy electrons (e-) to the electron transport chain (ETC) for the production of ATP and pyrimidine biosynthesis. Citrate transported into cytoplasm from the TCA cycle provides substrate for cytoplasmic acetyl- CoA production, necessary for fatty acid synthesis, which together with glycerol- 3 -phosphate generate lipids. Glucose-6-phosphate (glucose-6P) can alternatively be catabolized to ribose through the pentose phosphate shunt, which also generates NADPH for redox homeostasis. Glutamine is shown transported into the cell through ASCT2 and converted to glutamate by glutaminase (GLS), which is under the control of MYC through microRNA miR-23a/b. Glutamate is further catabolized to a-ketoglutarate (a-KG) for further oxidation in the TCA cycle. Inset: Schematic representation of the key metabolic pathways with M = malate shown in glutaminolysis. Figure IB is a schematic depicting a 3-D cutout of a tumor tissue block with a central capillary feeding an inner kernel of cells with oxygen and nutrients. This kernel uses oxidative phosphorylation with glucose and glutamine serving as substrates. As cell proliferative and are push away from the blood vessel, an oxygen gradient ((¾) is created with a concomitant increase in HIF-1 levels in the peripheral cuff of hypoxic cells, which utilizes glycolysis and perhaps glutaminolysis (conversion of glutamine to lactate). Note that lactate produced by LDHA in the hypoxic cuff is converted to pyruvate by LDHB in the central kernel of cells for oxidation in the mitochondrion (see Figure 1A). Figure 1C is a schematic depicting how microenvironment can cause tumor heterogeneity. Different levels of 0₂ and nutrients supplements can effect the microenvironment, including stroma and immune cells

Figure 2 depicts reprogramming of Glucose/Glutamine(GQ)-Metabolic Pathways by MYC and HIF-1. Schematic diagrams with changes in the key metabolic pathways depicted by the thickness of the arrows, resulting from MYC, HIF or both (with reference to Figure 1). Note that glutaminolysis involves the production of malate (M) from glutamine (Q) with subsequent conversion to lactate (L) via pyruvate (Py). G = glucose; G6P = glucose 6- phosphate; FA = fatty acid/lipid; R5P = ribose 5-phosphate; Nuc = nucleotide; aKG = alpha- ketoglutarate; E = glutamate.

Figures 3A-3C depict heterogeneity in the tumor microenvironment. Figure 3A is a photomicrograph of a histological section of a P493 human B lymphoma xenograft stained with an anti-body (brown) against pimonidazole (a hypoxia marker). The cartoon on the right depicts the cross-section of a blood vessel and the surrounding non-hypoxic and hypoxic cells. Further out from the blood vessel are necrotic cells. Figure 3B is a fluorescent micrograph of a histological section of a P493 xenograft labeled with pimonidazole (red) and BrdU (green). A conceptual diagram on the right depicts the hypothetical distribution of dividing (BrdU positive) cells in the inner cuff of cells around the blood vessel (red vertical line) and hypoxic cells in the outer kernel. Figure 3C are of images of histological sections of spleen, liver and a P493 lymphoma xenograft labeled with pimonidazole and stained (brown) via immunohistochemistry.

Figure 4 is a schematic of the HypoxCR lentiviral vector bearing bidirectional reporters for cell cycling (CMV-geminin-mCherry) and hypoxia (5xHRE-ODD-eGFP- PEST). Fluorescent micrograph on the right documents HEK 293T expressing the HypoxCR- egfp-mcherry vector imaged immediately after 24 hours of 2% oxygen (inset, right). Note the green hypoxic cells scattered with red cycling cells. Occasional hypoxic cycling (yellow) cells were seen.

Figure 5 is a schematic of the hypoxia response element (top) shown with hypoxic cells colored green to reflect the cells marked with the fusion protein of GFP-PEST. Transactivation by HIF-1 in response to hypoxia is shown (inset, right). Adapted from Urban Lendahl et al. 2009 Nature Review, Genetics.

Figure 6 depicts responses of hypoxia reporters. The panels on the left are fluorescent photomicrographs of 293T cells taken at the indicated period of normoxia after 24 hours of hypoxia. Schematic of the reporters are shown with 5xHRE driving a fusion protein comprising the HIF-1 oxygen dependent domain (ODD) fused to GFP or RFP and to a PEST sequence (from ODC) (upper, right). The immunoblots show the levels of either RFP or GFP after hypoxic cells were exposed to normal oxygen tension for the indicated times (lower, right). Tubulin served as a sample loading control.

Figure 7 is a schematic of the cell cycle (left) shown with cycling cells colored red to reflect the cells marked with the fusion protein of mCherry with geminin. A micrograph of cycling cells is shown with the flow cytometric histogram documenting the occurrence of red fluorescent cells in the S/G2M phases of the cell cycle (depicted far right). Adapted from Sakaue-Sawano et al. Chemistry & Biology 2008.

Figures 8A-8C depict responses of cell cycle reporters. Figure 8A depicts flow cytometry of hGem-GFP stably transfected Hela cells showing S-G2M phases with GFP as compared with the cell cycle distribution (determined by Hoechst stain) of the entire stably transfected population of cells. Figure 8B is a flow cytometric diagram representing 293T cells stably transfected with Geminin-hmAGl and stained with Hoechst (DNA content on the x-axis) (Middle). Cell cycle distribution (measured by Hoechst- staining for DNA content on the x-axis) of Geminin-hmAGl labeled cells that are non-fluorescent (non-green; 44% of total) or fluorescent (-56% of total). Figure 8C depicts micrographs of a monolayer of stably transfected 293T cells (phase; Right, top) with HypoCR (Geminin-hmAGl and HRE- ODD-mRFP-PEST) showing cycling fluorescent cells (Right, bottom).

Figures 9A and 9B depict hypoxic and cycling cells in 293T cell stably transfected with HypoxCR. Figure 9A is a superimposed micrograph (phase contrast and fluorescence) of a transformed focus of 293T cells stably labeled with HypoxCR (green = cycling; red = hypoxia). Figure 9 B depicts two-color flow cytometric diagrams (Right) illustrating the distribution of different subpopulations of HypcucCR-labeled 293T polyclonal cells in normoxia (left) or after 14 hours of 2% oxygen (right). Cycling cells labeled with Geminin- hmAGl fluoresces with intensities shown on the x-axis. The y-axis represents RFP fluorescence from the HREx5-ODD-mRFP-PEST vector. Bright hypoxic non-cycling cells (UL quadrant) were 5% and hypoxic cycling (UR quadrant) were 5%. Figures 10A and 10B depict the positions of insulator sequences relative to the reporter sequences of the bidirectional reporter cassette. Figure 10A is a schematic depicting the HRE-I-mCherry-Gemini vector which has an insulator sequence separating the bidirectional promoter sequences. Transcription of the promoters in different directions prevents interference of transcription of a reporter if both were oriented to transcribe in the same direction. Figure 10B is a schematic depicting the I-HRE-I-mCherry-Gemini-I vecotr which has an insulator sequences separating the bidirectional promoter sequences and flanking the bidirectional reporter cassette.

Figure 11 depicts crosstalk between bidirectional reporters. The position of the insulator in between the two reporters protects the GFP from continuous non-hypoxic expresssion.

Figure 12 depicts a potential consequence of having the dual promoters transcribing in the same direction.

Figures 13A and 13B depicts two constructs having insulators. Figure 13A depicts a construct having an insulator positioned at the 5' end of a first promoters that transcribes in the same direction as the second promoter (plvx-I-HRE-mcherry-Geminin). Figure 13B depicts a construct having an insulator positioned between two bidirectional promoters (plvx- HRE-I-mcherry- Geminin) .

Figure 14 depicts images of cells transfected with plvx-I-HRE-mcherry-Geminin under nomoxic and hypoxic conditions. Expression of GFP in cells exposed to hypoxic conditions was visualized at 0, 4, and 24 hrs after exposure to hypoxia.

Figure 15 depicts images of cells transfected with plvx-HRE-I-mcherry-Geminin under nomoxic and hypoxic conditions. Expression of GFP in cells exposed to hypoxic conditions was visualized at 0, 4, and 24 hrs after exposure to hypoxia.

Figure 16 depicts results of fluorescence activated cell sorting (FACS) of cells transfected with either plvx-THRE-mcherry-Geminin or plvx-HRE-I-mcherry-Geminin. FACS sorting of cells transfected with plvx-HRE-I-mcherry-Geminin (left) and with plvx-I- HRE-mcherry-Geminin (right).

Figure 17 depicts FACS sorting of cells transfected with plvx-HRE-I-mcherry- Geminin and exposed to normoxic conditions (left) or hypoxic conditions (right).

Figure 18 depicts FACS sorting of heterogeneous cells transfected with plvx-HRE-I- mcherry- Geminin .

Figure 19 is a schematic depicting the HRE-evoglow-TmCherry-Gemini vector which has evoglow sequences replacing the GFP sequences. Figure 20 depicts the increase dual sensitivity of the dual reporter vector expressing evoglow under normoxic conditions (left) and hypoxic conditions (0 hr, middle; 4 hr, right).

Figure 21 is a table of reporter plasmids and their properties.

Figures 22A-22D depict the 3-Dimensional reconstruction of HEK 293T tumor xenograft images of hypoxic and/or cycling cells marked with the HypoxCR lentiviral reporter. Figure 22A depicts a bidirectional reporter cassette for examining cell cycling (CMV-geminin-mCherry) and hypoxia (5xHRE-ODD-eGFP-PEST). Figures 22B-22D depict a 215 mm thick slice of a tumor cut 2mm from the surface is shown from different angles with a 450x450 mm window. Hypoxic cells are green and cycling cells are red.

Figures 23A-23D depict the effect of FXl 1 and Avastin on cycling and hypoxic cells.

Avastin reduced the percentage of cycling cells and FX11 reduced the percentage of hypoxic cells. Figure 23A depicts a bidirectional reporter cassette for examining cell cycling (CMV- geminin-mCherry) and hypoxia (5xHRE-ODD-eGFP-PEST). Figure 23B is an image of avastin treated cells, showing a reduction in cycling cells as visualized using the bidirectional reporter. Figure 23C is an image of FXl 1 treated cells, showing a reduction in hypoxic cells as visualized using the bidirectional reporter. Figure 23D is a chart showing percentages of red and green cells visualized with the bidirectional reporter in control cells and cells treated with Avastin, FX11, or Avastin and FX11.

Figure 24 depicts images showing FX11 treatment diminishes hypoxic regions (bright red) of P493 lymphoma. Rabbit anti-hypoxyprobe antibody was used as primary antibody. Texas-red anti-rabbit and DakoCytomation EnVision+ System-HRP anti-Rabbit were used as secondary antibodies for IF and IHC, respectively. Samples were analyzed under Axiovert 200 (Zeiss) fluorescence microscope at lOx magnification. Ten random fields from an untreated and a treated tumor were photographed.

Figure 25 depict exemplary evoglow, PEST, c-Myc, linker, GFP, HRE promoter,

HIF-1 ODD^530"603, HIF-1 ODD^400"620, insulator, insulator core (2x), mCherry, Gemini, FLAG polypeptide and nucleic acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions for detectably identifying cells as actively dividing and/or as hypoxic, and provides methods of using these compositions for the classification and isolation of said cells. Such compositions are further useful in methods of drug screening. As reported in more detail below, the present application provides expression vectors comprising a bidirection reporter cassette, wherein the cassette encodes at least two reporters, each of which is specifically expressed in cells that are actively cycling (e.g., CMV-geminin- mCherry) and in cells that are hypoxic (5xHRE-ODD-eGFP-PEST). The invention further provides methods for marking tumor cells and classifying them by phenotype, such that cells which are cycling are labeled with one detectable reporter (e.g., mCherry (red)) and those that are hypoxic are labeled with a second detectable reporter (e.g., green fluorescence protein (eGFP)). Desirably, the polynucleotide sequences encoding the detectable reporter polypeptides are separated by a polynucleotide sequence that insulates the two sequences ("an insulator polynucleotide"), such that their expression is independently regulated. In one particular example, the invention provides a lentiviral vector backbone comprising at least two polynucleotide sequences encoding a detectable molecule or reporter. The

polynucleotide reporter sequences are arranged bidirectionally. The expression vector further comprises a selectable marker (e.g., puromycin, hygromycin, or neomycin selectable markers).

The invention is based, at least in part, on the observation that tumor tissue is subject to pervasive hypoxia due to abnormal neo-vascularization, and that tumor tissue necessarily contains heterogeneous metabolic phenotypes. Tumor cells that are close to blood vessels are able to use glucose, glutamine and perhaps fatty acids as anabolic sources and energy derived from oxidative phosphorylation. As tumor cells distance from a blood vessel increases, oxygen tension is markedly diminished and such cells must undergo hypoxic energy metabolism or rapidly die.

The present invention provides cell cycling and hypoxia reporters that provide for the identification of subpopulations of tumors cells marked by these reporters. Such cells can be isolated by phenotype and analysed to determine those factors that permit their survival within the heterogenous tumor environment. Once isolated, these tumor subfractions may be used to identify therapeutics that target glycolysis or mitochondrial function. In other embodiments, these subpopulations are used to characterize standard chemo therapeutics to understand how the tumor microenvironment and cancer metabolism influence therapeutic responses to different categories of drugs. These screening approaches will identify combination therapies combining standard chemotherapeutic agents with novel agents that target metabolism to eliminate different subpopulations of presumably monoclonal cancer cells in the tumor tissue. Cell Cycling Reporter

A cell cycling reporter of the invention is selectively expressed in cells that are actively dividing, thereby providing for their identification. This expression is distinct and distinguishable from the expression of a hypoxic reporter polypeptide in cells that are hypoxic. Advantageously, as reported herein, a cell cycling reporter polypeptide (e.g., fluorescent protein mCherry or any other detectable protein known in the art) is fused with a moiety that destabilizes the reporter polypeptide, to limit its half-life. Such a moiety is referred to herein as a "destabilizing moiety." Preferably, the half-life of the reporter polypeptide fused to the destabilizing moiety is equal to about the length of time required for a cell to divide. Typically, a cell divides every twenty-four hours, more or less (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 hours). Thus, the destabilizing moiety provides for the breakdown or inactivation of the reporter polypeptide within a time frame that is approximately equal to about the time required for cell division. Exemplary destabilizing moieties include, but are not limited to, a PEST sequence, a fragment of geminin (e.g., a 110 amino acids of geminin), ubiquitin, or any other moiety that provides for the selective degradation or inactivation of the reporter polypeptide. In one embodiment, the protein is selectively degraded during the Gl-phase of the cell cycle. In one particular embodiment, a detectable fusion protein is linked to 110 amino acids of geminin, which stabilizes the protein during the S/G₂M phases of the cell cycle. In another embodiment, the moiety that provides for the selective degradation of the protein comprises optimized human codons.

Hypoxia Reporter

A hypoxia reporter of the invention is selectively expressed in cells that are hypoxic, thereby providing for their identification. The expression of the hypoxia reporter polypeptide is distinct and distinguishable from the expression of the cell cycling reporter polypeptide. Any polynucleotide that is selectively expressed in a hypoxic cell type may be used. For example, one or more hypoxia response element may be fused to a polynucleotide sequence encoding a detectable reporter, thereby providing for the expression of a detectable reporter polypeptide in a hypoxic cell. If desired, the hypoxia reporter comprises at least about three, four, five, six, seven, or eight hypoxia response elements (HREs). In one particular embodiment, five HREs drive the expression of a cDNA encoding a fusion of the HIF-1 oxygen dependent domain (ODD) with eGFP. One of skill in the art will appreciate that any detectable polypeptide known in the art may be used. Preferably, this reporter polypeptide is fused to a detabilizing moiety, such as a PEST sequence (e.g., the ornithine decarboxylase (ODC) PEST sequence). Preferably, this reporter construct is designed to provide a robust hypoxic response, but with a fluorescent protein that has a half-life which is shorter than one cell cycle time. As reported in detail below, a hypoxia-induced eGFP fusion protein lasting less than 8 hours in normoxic conditions is used. In other embodiments, the ODD and PEST sequences function to ensure that the half-life is between 1 and 5 hours (e.g., ~4 hours), between 3-6 hours, or between 5-10 hours. In other embodiments, the half-life of the hypoxia reporter polypeptide is 1, 2, 3, 4, 5, 6, 7, 8, or 9 hours. Without the ODD and PEST sequences, eGFP fluorescence was observed 48 hours after re-oxygenation.

As reported herein, HEK 293T cells were infected with viral particles containing the

HypoxCR reporter, for study in vitro and in situ as xenografts. One of skill in the art will appreciate that virtually any type of cell can be analyzed using the constructs of the invention. Desirably, the cells and cell lines disclosed herein are engineered to express an expression vectors described herein. Typically, an expression vector is used to transfect the cells. The term "transfection" as used herein means an introduction of a foreign DNA or RNA into a cell by mechanical inoculation, electroporation, infection, particle bombardment,

microinjection, or by other known methods. Alternatively, one or a combination of expression vectors can be used to transform the cells and cell lines. The term

"transformation" as used herein means a stable incorporation of a foreign DNA or RNA into the cell which results in a permanent, heritable alteration in the cell. A variety of suitable methods are known in the field and have been described. See e.g., Ausubel et al, supra;

Sambrook, supra; and the Promega Technical Manual.

In particular invention embodiments, a cell or cell line of choice is manipulated so as to be stably transformed by an expression vector of the invention. However, for some invention embodiments, transient expression of the vector (e.g., for less than about a week, such as one or two days) will be more helpful. Cells and cell lines that are transiently transfected or stably transformed by one or more expression vectors disclosed herein will sometimes be referred to as "recombinant". By the phrase "recombinant" is meant that the techniques used for making cell or cell line include those generally associated with making and using recombinant nucleic acids (e.g., electroporation, lipofection, use of restriction enzymes, ligases, etc.).

As discussed herein, this also relates to methods for detecting and in some cases analyzing agents that alter expression of a cell cycling reporter, a hypoxic reporter, or that reduce the survival or proliferation of a neoplastic cell (e.g., tumor cell) that expresses one or both of these reporters. Certain of those agents can be further selected if needed to identify those with therapeutic capacity to selectively induce the cell death of a cell expressing a hypoxia reporter, a cell cycling reporter, or both. Preferred detection and analysis methods include both in vitro and in vivo assays to determine the therapeutic capacity of agents to prevent, treat, prolong the onset of, or help alleviate the symptoms of a neoplasm (e.g., tumor).

Polynucleotide Delivery

Nucleic acid molecules encoding detectable polypeptides of the invention can be delivered to cells (e.g., neoplastic cells, tumor cells). The nucleic acid molecules must be delivered to the cells of a subject in a form in which they can be taken up so that

therapeutically effective levels of a reporter protein can be produced. Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used py, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, a polynucleotide encoding a reporter protein, variant, or a fragment thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980- 990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S- 83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No.

5,399,346). Most preferably, a viral vector is used to administer an expression vector of the invention to a target cell, tumor tissue, or systemically. In one preferred embodiment, a VLP is used to deliver an expression vector of the invention as delineated in the Examples provided herein below. Non- viral approaches can also be employed for the introduction of a therapeutic to a cell (e.g., a tumor cell or neoplastic cell). For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid molecule in the presence of lipofectin (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247:1465, 1990). Preferably the nucleic acids are administered in combination with a liposome and protamine.

Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell.

Expression of a reporter construct of the invention can be directed from any suitable promoter and regulated by any appropriate mammalian regulatory element (e.g., hypoxia response element). If desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as hypoxia responsive elements, HIF-1 oxygen dependent domain, and tissue- or cell-specific enhancers. Alternatively, regulation can be mediated by cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above. Reporter Polypeptides and Analogs

The invention provides for the expression of an expression vector comprising detectable reporter polypeptides that classify cells as cycling, hypoxic, or, more rarely, as cycling and hypoxic. The expression vectors of the invention encode at least two

bidirectionally linked reporter polypeptides, each of which is fused to a destabilizing moiety. One of the detectable reporters is selectively expressed in cycling cells and the other is expressed in hypoxic cells. In general, the reporters are separated by an insulator sequence that provides for the independent regulation of the reporters. The insulator sequence may vary widely in length. Desirably, an insulator polynucleotide is of a length sufficient to optimize the independent expression of the polynucleotide sequences that it separates. In one embodiment, the insulator is between about 100 and 2000 nucleic acids in length (e.g., 100, 200, 250, 500, 750, 1000, 1250, 1500, 1750, 2000) or even longer. In other embodiments, the insulator is between 1 and 2000bp in length, preferably about 1250 bp. In certain

embodiments, a shorter insulator sequence is used to minimize the size of the polynucleotide comprising the reporter and insulator sequences (e.g., a viral expression vector comprising an insert above 7kb reduces the packaging efficiency into virus). If desired, the reporter constructs are flanked at either end with insulator sequences, example, an insulator sequence separates the regulatory elements controlling expression of the cell cycling reporter or the hypoxic reporter from upstream/downstream sequences within the vector backbone. In one embodiment, a reporter cassette comprises an insulator located between the two reporters to prevent interference between two promoters. In one embodiment, a reporter cassette comprises an insulators flanking the downstream ends of a reporter. In one embodiment, a reporter cassette comprises two insulators flanking the downstream ends of both reporters and an insulator located between the two reporters to prevent the interference between two promoters.

Also included in the invention are reporter polypeptides, destabilizing moieties, or fragments thereof that are modified in ways that desirably alter them. In one embodiment, the invention provides methods for optimizing a reporter amino acid sequence or nucleic acid sequence by producing an alteration in the sequence. Optimization of codons for expression in a human cell is useful for the expression of a nucleic acid sequence that is not based on human nucleic acid sequence, e.g., an endogenous human gene. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications. These modifications may be made in either the regulatory regions (e.g., hypoxia response elements, ODD), in the detectable reporters, or in the destabilizing moieties. In one embodiment, the detectable reporter or destabilizing moiety is a detectable reporter or destabilizing moiety analog. In one embodiment, these alterations are made to enhance expression of the sequence in a mammalian cell.

In other embodiments, the invention further includes analogs of any polypeptide of the invention. Analogs can differ from a naturally occurring polypeptide of the invention by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally occurring amino, acid sequence of the invention. The length of sequence comparison is at least 5, 10, 15 or 20 amino acid residues, preferably at least 25, 50, or 75 amino acid residues, and more preferably more than 100 amino acid residues.

In various embodiments, the reporter polypeptides and destabilizing moieties of the invention are altered to delete, substitute, or modify amino acid residues that alters the detectable characteristics of the reporter polypeptide or that alters the half-life of the reporter polypeptide. Screening methods to identify polypeptides fused to destabilizing moieties having the desired half-life are known in the art and are described herein in the Examples.

Analogs can differ from the naturally occurring polypeptides of the invention by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to

ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). A "detectable reporter" is a polypeptide that comprises a moiety that renders it detectable, via any means, including spectroscopic, photochemical (e.g., luciferase, GFP), biochemical, immunochemical, or chemical means. For example, useful labels include fluorescent dyes, such as GFP, RFP, Evoglow, and mcherry. GFP derivatives have been engineered with useful properties (e.g., different emission spectra, increased fluorescence, photo stability) and include, for example, blue fluorescent protein (BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (CFP, ECFP, Cerulean, CyPet), and yellow fluorescent protein (YFP, Citrine, Venus, YPet) (Shaner et al. (2005). "A guide to choosing fluorescent proteins" Nat Methods 2 (12): 905-9). Cells labeled with fluorescent labels can be selected and separated using routine methods, including fluorescence activated cell sorting (FACS) or Laser-Enabled Analysis and Processing (LEAP) microplate-based cytometry. Other detectable labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, electron-dense reagents, enzymes (e.g., horseradish peroxidase, alkaline

phosphatase), biotin, digoxigenin, or haptens.

Nucleic Acid Molecules Encoding Cell Cyling or Hypoxia Reporter Polypeptides

The invention further includes nucleic acid molecules that encode a reporter polypeptide. Particularly useful in the methods of the invention are nucleic acid molecules encoding a mCherry reporter polypeptide, GFP polypeptide, or fragments thereof. The sequence of exemplary nucleic acid molecules are provided herein. Reporter Polypeptide Expression

In general, reporter polypeptides of the invention may be produced by transformation of a suitable host cell with all or part of an expression construct of the invention. Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used. The precise host cell used is not critical to the invention. A host cell is any cell (e.g., eukaryotic cell) that contains an expression vector.

A polypeptide of the invention may be produced in a eukaryotic host cell (e.g., a mammalian cells, e.g., NIH 3T3, HeLa, or preferably COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al., Current Protocol in Molecular Biology, New York: John Wiley and Sons, 1997). The method of transformation or transfection and the choice of expression vector can be selected to optimize the identification of cycling and hypoxic cells within a mixed population. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).

A variety of expression systems exist for the production of the polypeptides of the invention. Expression vectors useful for producing such polypeptides include, without limitation, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculo viruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof. In one particular embodiment, the invention provides a lentiviral vector backbone comprising one or more polynucleotides encoding reporter constructs described herein. An expression vector is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a particular gene in a host cell. Typically, gene expression is placed under the control of certain regulatory elements (e.g., hypoxia responsive elements, ODD elements). Other regulatory elements include constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers. The invention provides for the expression of any of detectable polypeptides described herein via an expression vector. The sequence of exemplary expression vectors are provided herein. In addition, the invention features host cells (e.g., mammalian, rodent, human cells) comprising a nucleic acid sequence that encodes any reporter polypeptide described herein. In another approach, an expression vector of the invention is expressed in a transgenic organism, such as a transgenic animal. By"transgenic"is meant any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell, or part of a heritable extra chromosomal array. As used herein, transgenic organisms may be either transgenic vertebrates, such as domestic mammals (e. g. , sheep, cow, goat, or horse), mice, or rats. In one embodiment, the reporter constructs of the invention are expressed in a transgenic animal, such as a rodent (e.g., a rat or mouse). In addition, cell lines from these mice may be established by methods standard in the art. Construction of transgenes can be accomplished using any suitable genetic engineering technique, such as those described in Ausubel et al. (Current Protocols in

Molecular Biology, John Wiley & Sons, New York, 2000). Many techniques of transgene construction and of expression constructs for transfection or transformation in general are known and may be used for the disclosed constructs.

Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Taconic (Germantown, N.Y.). Many strains are suitable, but

Swiss Webster (Taconic) female mice are desirable for embryo retrieval and transfer. B6D2F (Taconic) males can be used for mating and vasectomized Swiss Webster studs can be used to stimulate pseudopregnancy. Vasectomized mice and rats are publicly available from the above-mentioned suppliers. However, one skilled in the art would also know how to make a transgenic mouse or rat. An example of a protocol that can be used to produce a transgenic animal is provided below.

Production Of Transgenic Mice And Rats

The following is but one desirable means of producing transgenic mice. This general protocol may be modified by those skilled in the art.

Female mice six weeks of age are induced to superovulate with a 5 IU injection (0.1 cc, IP) of pregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours later by a 5 IU injection (0.1 cc, IP) of human chorionic gonadotropin (hCG, Sigma). Females are placed together with males immediately after hCG injection. Twenty-one hours after hCG injection, the mated females are sacrificed by CO. sub.2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts and placed in Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin (BSA, Sigma). Surrounding cumulus cells are removed with hyaluronidase (1 mg/ml). Pronuclear embryos are then washed and placed in Earle's balanced salt solution containing 0.5% BSA (EBSS) in a 37.5.degree. C. incubator with humidified atmosphere at 5% CO. sub.2, 95% air until the time of injection. Embryos can be implanted at the two-cell stage.

Randomly cycling adult female mice are paired with vasectomized males. Swiss Webster or other comparable strains can be used for this purpose. Recipient females are mated at the same time as donor females. At the time of embryo transfer, the recipient females are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5% avertin per gram of body weight. The oviducts are exposed by a single midline dorsal incision. An incision is then made through the body wall directly over the oviduct. The ovarian bursa is then torn with watchmakers forceps. Embryos to be transferred are placed in DPBS

(Dulbecco's phosphate buffered saline) and in the tip of a transfer pipet (about 10 to 12 embryos). The pipet tip is inserted into the infundibulum and the embryos are transferred. After the transferring the embryos, the incision is closed by two sutures.

A desirable procedure for generating transgenic rats is similar to that described above for mice (Hammer et al., Cell 63:1099-112, 1990). For example, thirty-day old female rats are given a subcutaneous injection of 20 IU of PMSG (0.1 cc) and 48 hours later each female placed with a proven, fertile male. At the same time, 40-80 day old females are placed in cages with vasectomized males. These will provide the foster mothers for embryo transfer. The next morning females are checked for vaginal plugs. Females who have mated with vasectomized males are held aside until the time of transfer. Donor females that have mated are sacrificed (CO. sub.2 asphyxiation) and their oviducts removed, placed in DPBA

(Dulbecco's phosphate buffered saline) with 0.5% BSA and the embryos collected. Cumulus cells surrounding the embryos are removed with hyaluronidase (1 mg/ml). The embryos are then washed and placed in EBSs (Earle's balanced salt solution) containing 0.5% BSA in a 37.5. degree. C. incubator until the time of microinjection.

Once the embryos are injected, the live embryos are moved to DPBS for transfer into foster mothers. The foster mothers are anesthetized with ketamine (40 mg/kg, IP) and xulazine (5 mg/kg, IP). A dorsal midline incision is made through the skin and the ovary and oviduct are exposed by an incision through the muscle layer directly over the ovary. The ovarian bursa is torn, the embryos are picked up into the transfer pipet, and the tip of the transfer pipet is inserted into the infundibulum. Approximately 10 to 12 embryos are transferred into each rat oviduct through the infundibulum. The incision is then closed with sutures, and the foster mothers are housed singly. Screening Assays

As detailed herein, the invention provides for compositions that specifically identify tumor cells that are cycling and allow them to be distinguished from those cells that are hypoxic. In one embodiment, a cell cycling reporter comprises a fusion of the fluorescent protein mCherry with 110 amino acids of geminin, a protein that is selectively degraded in the Gl -phase of the cell cycle. This fusion protein is used to marks cells that are cycling and dividing with mCherry. A hypoxia reporter comprises five hypoxia response elements (HREs) driving a cDNA encoding the fusion of the HIF-1 oxygen dependent domain (ODD) with eGFP and the ornithine decarboxylase (ODC) PEST sequence is used to mark cells that are hypoxic (i.e., that express the reporter only under hypoxic conditions, and that fail to detectably express the reporter under normoxic conditions. Cells expressing the constructs of the invention may be used for in vitro analysis and in situ analysis as xenografts.

In particular embodiments, cells, tissues, and animals comprising expression vectors delineated herein are useful for the high-throughput low-cost screening of candidate compounds. In a conventional approach, chemotherapeutic agents that reduce the survival or proliferation of cancer cells are screened in vitro under normoxic conditions. Agents selected in such conventional screening approaches often fail to deliver the promised therapeutic results when they are tested in animal models in vivo or in clinical trials. The reason that such agents fail, is often difficult to determine. The present invention provides improved methods for characterizing the effects of chemotherapeutic agents on tumors cells. In particular, it provides for the phenotypic classification of cells comprising expression vectors of the invention. Cells that are refractory to the effects of a candidate chemotherapeutic agent can be classified as undergoing cell cycling or by their response to oxygen.

In one exemplary approach, a xenograft that expresses an expression vector delineated herein is contacted with a chemotherapeutic agent that is expected to reduce the proliferation or survival of the cells. After exposure to the agent, any remaining viable cells are assayed for expression of the reporter constructs. This allows for the phenotypic classification of the surviving refractory cells. For example, where the remaining viable cells express the hypoxic reporter construct this suggests that hypoxic cells are less susceptible to the chemotherapeutic effects of the agent than other cells. If desired, the remaining cells are screened against candidate agents in a second screen to identify agents that selectively reduce the survival or proliferation of refractory hypoxic cells. Agents identified in the second screen are useful for reducing the survival or proliferation of the refractory hypoxic cells. Such agents may be used alone or in combination with conventional chemotherapeutic agents. If desired, refractory cells are isolated and used to establish cell lines, or are genomically or proteomically characterized. Screening assays are described for example in Le et al., Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression. Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):2037-42.

The effect of candidate compounds on cell survival may be assessed in vitro in. In one working example, candidate compounds are added at varying concentrations to the culture medium of cultured cells maintained under normoxic (e.g., 6% oxygen), hyperoxic (e.g., about 20% oxygen, and/or hypoxic (e.g., about 1%, 2%, 3%, 4%, 5%) conditions.

Methods for culturing cells under such conditions are known in the art, and include for example, the use of microcarrier beads to culture transformed cells which grow as multilayers as compared to non-transformed cells that undergo contact inhibition and NanoCulture Plate (NCP from Scivax - www.scivax.com) for spheroid cultures. Such methods are described for example in Loessner et al. Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells. Biomaterials. 2010 Nov;31(32):8494-506. Cell survival is then measured using standard methods. In one example, the level of cell death or apoptosis in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate molecule. A compound that promotes an increase in apoptosis, or a reduction in cell proliferation of hypoxic cells, cycling cells, or both, is considered useful in the invention; such a candidate compound may be used, for example, as a therapeutic to prevent, delay, ameliorate, stabilize, or treat a neoplasia or cancer. Detectable reporters are chosen that emit different wavelengths of light, such that the hypoxic reporter may be differentiated from the cycling reporter.

Compounds isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography). Compounds isolated by this approach may also be used, for example, as therapeutics to treat any disease or condition characterized by excess cell death in a subject. A "subject" is typically a mammal in need of treatment, such as a human or veterinary patient (e.g., rodent, such as a mouse or rat, a cat, dog, cow, horse, sheep, goat, or other livestock). Test Compounds and Extracts

In general, agents are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries, according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Compounds used in screens may include known compounds (for example, known therapeutics used for other diseases or disorders). Alternatively, virtually any number of unknown chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds.

Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, chemical compounds to be used as candidate compounds can be synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds identified by the methods described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909, 1993; Erb et al, Proc. Natl. Acad. Sci. USA 91:11422, 1994;

Zuckemiann et al, J. Med. Chem. 37:2678, 1994; Cho et al, Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al, J. Med. Chem. 37:1233, 1994. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al, Proc Natl Acad Sci USA 89:1865-1869, 1992) or on phage (Scott and Smith, Science 249:386-390, 1990; Devlin, Science 249:404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382, 1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra.).

In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity should be employed whenever possible.

When a crude extract is found to selectively reduce the proliferation of a cell expressing the hypoxic reporter, of a cell expressing the cycling reporter, or both, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that reduces the survival or proliferation of a neoplastic cell (e.g., tumor cell). Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful as therapeutics for the treatment of any disease or condition associated with unregulated proliferation.

The practice of the present invention employs, unless otherwise indicated,

conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,

"Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989);

"Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987);

"Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,

"Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989);

"Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987);

"Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

Example I. Metabolic Pathways and Tumor Microenvironment in Cancer.

The Warburg effect, which describes the propensity for cancer cells and tissues to take up glucose avidly and convert it almost exclusively to lactate (aerobic glycolysis), has been an important tenet of cancer cell metabolism ^{1 5}. The importance of aerobic glycolysis is illustrated clinically by the distinct phenotype of high glucose uptake documented by positron emission tomography (PET) scanning of human cancers with radiolabeled 2- deoxyglucose, and molecularly by the de -regulation of oncogenes and tumor suppressors that result in cell autonomous changes promoting the conversion of glucose to lactate. However, not all cancers are PET-positive, and not all models of neoplastic transformation are associated with increased aerobic glycolysis; in fact, a few have increased mitochondrial function ⁶. The prevailing model of cancer metabolism is based largely on studies from in vitro tissue culture conditions at 20% oxygen, which do not reflect the hypoxic conditions present within a tumor. Along with previous studies documenting that Myc induces mitochondrial biogenesis, recent findings that glutamine catabolism is stimulated by Myc must be incorporated into current models of cancer metabolism, particularly if these alterations are to be exploited for therapeutic purposes 7-^"10.

Metabolic Pathways in Cancer. The Warburg effect describes the high flux of glucose through glycolysis, which converts hexoses to trioses. The trioses are a source of glycerol for lipid synthesis and of carbons for conversion to pyruvate for the production of ATP. Pyruvate is then converted to lactate ^{1 5}, which results in a high output of lactate even with adequate oxygen levels (Figure 1A). Glucose may also be catabolized through the pentose phosphate pathway to generate NADPH for redox homeostasis and ribose for nucleotide biosynthesis. In the presence of oxygen, pyruvate can be converted by pyruvate dehydrogenase (PDH) to acetyl-CoA for further oxidation through the tricarboxylic acid (TCA) cycle. The conversion of pyruvate to acetyl-CoA is blocked when pyruvate dehydrogenase kinase 1 (PDK1) is up-regulated. The PDK1 kinase is induced under hypoxic conditions by the hypoxia inducible transcription factor HIF-1 and functions to phosphorylate and inactivate PDH ^u. Less well characterized as an energy source and anabolic source of carbon and nitrogen is glutamine (Figure 1A), the amino acid with the highest circulating concentration in human blood. Glutamine is taken up by cells and may be utilized as an amino acid for protein synthesis, although it is primarily converted to glutamate by glutaminase. Glutamate is then converted to a-ketoglutarate, an oxidative substrate for the TCA cycle. Glutamine can also be converted to pyruvate and then to lactate through malate, a TCA cycle metabolite of a-ketoglutarate, in a less well understood process termed glutaminolysis (Figure 2). Thus, glutamine can serve as an important source of cellular energy and anabolic carbon and nitrogen.

Many studies of oncogenic alterations of metabolism have suggested that cell autonomous changes due to the activation of oncogenes or loss of tumor suppressors are key drivers for the high conversion of glucose to lactate. These include early studies that documented the association of Src- and Ras-transformation with increased glucose transporter expression and our study demonstrating the direct trans-activation of the LDHA

(lactate dehydrogenase A) gene by the Myc onco-protein (Figure 1A) 12 ' 13. Later studies linked Ras, VHL, and mutations of isocitrate dehydrogenase 1 (IDH1), succinate dehydrogenase (SDH), and fumarate hydratase (FH) to the activation of HIF-1, even in normoxia, which in turn induces glycolytic enzyme gene expression ^{1 4}'⁵'¹⁴. Furthermore, the Akt oncogene was shown to stimulate glycolysis post-transcriptionally, and the p53 tumor suppressor emerged as another regulator of mitochondrial function and glycolysis, such that loss of p53 is associated with enhanced glycolysis 2 ' 4. Collectively, these observations support the idea that activation of oncogenes and loss of tumor suppressors result in the induction of cell autonomous aerobic glycolysis, independent of hypoxia, or the Warburg effect. However, there are a number of observations that are not encompassed by our current model of cancer metabolism.

Reprogramming of Glucose-Glutamine (GQ) Metabolic Pathways by Myc and HIF- 1. Previous models of cancer metabolism were largely based on studies from in vitro tissue culture conditions, which do not recapitulate the hypoxic condition present within a tumor ^15~

18 (Figures IB, 1C). Tumor cell adaptation and tolerance of hypoxia are critical features of a robust cancer cell. Compared to most normal tissues, which are well-oxygenated, tumor tissues are generally hypoxic with some more oxygenated areas around poorly-formed tumor blood vessels (Figure 2) 18-^"20. While constitutive cell autonomous changes favoring aerobic glycolysis could be advantageous to cancer cells under certain conditions, the presence of tumor hypoxia indicates that adaptive changes are also important, when HIF-1 is not constitutively activated. For example, the activation of HIF-1 in hypoxic conditions not only induces an adaptive metabolic program, but also triggers angiogenesis 21. In tumors with constitutive activation of HIF-1 downstream of altered oncogenes or tumor suppressors, the adaptive response to hypoxia may not be as relevant - but how constitutive HIF-1 expression, which inhibits normal cell proliferation, permits continued neoplastic cell proliferation remains poorly understood. Hypoxia, putatively through HIF-1, induces the expression of cyclin dependent kinase inhibitors (CDKIs) p21 and p27 in normal cells, while ectopic expression Myc is documented to inhibit the expression or function of these CDKIs 22-^"33 . The interplay between HIF and Myc, hence, depends on whether the MYC gene is deregulated or remained susceptible to inhibitory circuitries that are activated by hypoxia in non-transformed cells.

Additionally, the commensal relationships between hypoxic and non-hypoxic cells in the tumor tissue are important for tumor maintenance and neo-vascularization. The importance of heterogeneity within a tumor bed and symbiosis between tumor cells was recently illustrated by the documentation that hypoxic tumor cells produce lactate, which can be re-cycled and re-utilized as pyruvate for oxidative phosphorylation by the more oxygenated tumor cells (Figure IB) 34 ' 35. Additional studies also suggest a symbiotic metabolic relation between tumor and stromal cells. Compared to human lung cancer cells, which express high levels of HIF-1, LDHA and PDK1, the accompanying tumor associated stromal fibroblasts have decreased PDK1 and increased PDH. This suggests a commensal relation between tumor and stroma, in which stromal cells may not only undergo oxidative phosphorylation but also recycle lactate released from tumor cells ³⁶. Without being bound to a particular theory, the microenvironmental heterogeneity of tumor cells, in the backdrop of findings that Myc can stimulate mitochondrial biogenesis, oxygen consumption, and glycolysis, suggests that MYC drives both aerobic glycolysis and oxidative phosphorylation when oxygen is ample in tumor cells located immediately around a blood ves sel 37. When oxygen is limited in cells located distal to the blood vessel, deregulated Myc collaborated with HIF-1 to attenuate mitochondrial respiration, but not necessarily other mitochondrial biosynthetic functions, and to increase glycolysis for adaptation to the tumor

microenvironment (Figure 2). Myc induces mitochondrial biogenesis in proliferating cells while inhibiting mitochondrial respiration with HIF-1 under hypoxic conditions.

Mitochondria not only provide a means for efficient production of ATP in the presence of oxygen, but they also serve as a factory for many other building blocks of a growing cell. These building blocks include pyridimines, whose synthesis is strictly linked to the electron transport chain via the activity of dihydro-orotate dehydrogenase, the carbon backbone for amino acids, as well as citrate which is extruded into the cytoplasm and converted to acetyl- CoA for lipid biosynthesis (Figures 1A and 2) ^{1 5}. The stimulation of glucose uptake and metabolism by Myc, on the other hand, provide carbon backbone for critical cellular processes, such as ribose for nucleotide biosynthesis and NADPH through the pentose phosphate pathway for redox homeostasis, triglycerides and ATP through glycolysis.

Because Myc induces mitochondrial biogenesis whether Myc could also affect the composition of the mitochondria and alter their function was determined. To this end, high- resolution 2-D gel electrophoresis of purified mitochondria from human B lymphocytes with low Myc or high Myc expression was performed . Mitochondrial glutaminase (encoded by GLS) was among seven proteins identified by mass spectrometry as being highly induced by Myc. This enzyme catalyzes the conversion of glutamine to glutamate, which can then be converted to a-ketoglutarate as a substrate for the TCA cycle. Because of this observation, genes involved in glutamine catabolism were examined. ASCT2 (or SLC5A1) and SLC7A1, both involved in glutamine transport, were found to behave as direct Myc target genes (Figure 1A). Given that glutamine influx could be increased by Myc, it is notable that glutamine is not only an energetic and anabolic substrate, but also provides nitrogen for amino acid and nucleic acid biosynthesis and precursors for glutathione synthesis for redox homeostasis (Figures 1A and 2). GLS protein levels robustly responded to Myc, but its mRNA levels did not vary significantly with changes in Myc levels in a human B cell line. Without being bound to a particular theory, we hypothesized that Myc regulates GLS at the post-transcriptional level. Myc directly repressed expression of microRNAs miR-23a and miR-23b, which target the 3'-UTR seed sequence in the GLS transcript to down-regulate its translation (Figure 1A). It is notable that GLS transcript levels might also be affected by miRNAs, particularly since GLS mRNA levels vary with Myc in fibroblasts (19). These observations, along with early observations that glutamine deprivation of Myc-transformed human cells triggered apoptosis (20), indicate that Myc stimulation of glutamine catabolism is part-and-parcel of its central role in integrating cell proliferation with metabolism.

Cancer in the Tumor Microenvironment. If Myc stimulates glutamine oxidation and mitochondrial biogenesis as well as glycolysis, then how does aerobic glycolysis and oxidative phosphorylation participate in tumorigenesis and tumor maintenance? Inducible- MYC human B cell lymphoma model was discovered to consume oxygen and glutamine in the presence of oxygen 7 ' 37. When oxygen is deprived, however, Myc collaborated with HIF- 1 to increase glycolytic enzyme gene expression and attenuated the conversion of pyruvate to acetyl-CoA by PDH through the induction its negative-regulatory kinase PDK1 (Figures IB and 2) 11 '38. Extant data supported the existence of the conversion of glutamine to lactate via glutaminolysis which uses half of the TCA cycle, suggesting glutaminolysis may play a role in hypoxic cells (Figure 2). Nonetheless, without being bound to a particular theory, the ability of Myc to stimulate oxidative phosphorylation and glycolysis simultaneously suggested that it offers an advantage to cancer cells in the tumor tissue microenvironment which consists of both hypoxic regions and more oxygenated regions around blood vessels (Figures IB, 1C, and 2).

In this regard, re-thinking cancer metabolism must consider the cellular heterogeneity within a tumor, such that Myc confers a growth advantage to tumor cells with adequate oxygen both by increasing mitochondrial biogenesis and glutamine metabolism for use in ATP production, anabolic carbon and nitrogen sources, and redox homeostasis, and by increasing glucose flux which provides anabolic carbons for ribose and fatty acid

biosynthesis, and produces NADPH (Figure 2). Without being bound to a particular theory, cells that consume oxygen and proliferate around the tumor blood vessels are hypothetically pushed into hypoxic regions, which are caused by oxygen consumption by the remaining non-hypoxic cells around blood vessels. The hypoxic cells then induce HIF-1 and adapt or die, unless they already have constitutive expression of HIF-1 which should provide a growth advantage (Figures IB and 2). While hypoxia normally triggers cell cycle arrest in non- transformed cells (except endothelial cells), deregulated MYC suppresses the cyclin dependent kinase inhibitors p21 and p27, permitting continued cell proliferation in hypoxia, when nutrients and the energy supplies are adequate 22-^"33. Without being bound to a particular theory, the model proposed herein suggests that the ability of Myc to collaborate with HIF-1 confers a metabolic advantage by inducing high fluxes of glucose through glycolysis, which must be accompanied by a replenishable nitrogen source of substrate for continued nucleotide biosynthesis (Figure 2). With severe hypoxia or near anoxia, cancer cells arrest in S-phase, cease to proliferate, adapt (or die) and allow for the ensuing angiogenesis triggered by both HIF-1 and Myc to replenish nutrients and oxygen 22.

Autophagy may also play a role for survival in this model. However, once the new sources of nutrients are available, the cells could resume proliferating with total disregard to normal external cues.

In this regard, Myc and HIF-1 are both critical tumor maintenance factors, whose target genes can be exploited for therapeutic purposes. LDHA, a transcription target gene common to Myc and HIF-1, is necessary for the transformation phenotype in vitro and tumor maintenance in vivo and is hence an attractive therapeutic target. However, re-thinking cancer metabolism in the context of the tumor tissue suggests that combinations of multiple agents affecting glycolysis, glutamine, or both may be necessary for the effective targeting of tumor metabolism for cancer therapy.

An picture of tumor metabolism is emerging that transcends cell autonomous changes documented largely in vitro under 20% oxygen to the reality of the tumor tissue that has pervasive hypoxia due to abnormal neo-vascularization. Without being bound to a particular theory, a model for Myc-driven cancers is proposed in which the tumor tissue contains heterogeneous metabolic phenotypes (Figure IB). Cells transformed by MYC nearby a blood vessel would be able to use glucose, glutamine and perhaps fatty acids as anabolic sources and energy derived from oxidative phosphorylation (Figure 2). These dividing cells multiply and expand away from the blood vessel to the point where oxygen tension is markedly diminished through consumption by cells around the blood vessel. The hypoxic Myc- transformed cells with the aid of HIF activation could then enhance glycolysis or the conversion of glucose to lactate. Glutaminolysis or the conversion of glutamine to lactate (rather than glutamine oxidation) may play a role in hypoxic energy metabolism. Nonetheless, it is hypothesized that lactate produced by the hypoxic Myc-driven cancer cells could be recycled to pyruvate for oxidation by tumor cells located immediately around a blood vessel (Figures IB and 2).

This model can be studied directly by the tools engineered and described herein. The development of fluorescent reporters can be used to identify cells that are cycling and proliferating and those that are hypoxic. With these tools, genomic responses of cycling and hypoxic subpopulations of tumors cells marked by the reporters can be studied. Furthermore, key nodal points downstream of HIF-1 and Myc can be studied in cycling and hypoxic subpopulations Using reporters that identify cycling and hypoxic cells, the response of tumors to standard chemo therapeutics can be assessed to understand directly how the tumor microenvironment and cancer metabolism influence therapeutic responses to different categories of drugs. Furthermore, the responses of these tumor subfractions to therapeutics that target glycolysis or mitochondrial function can be characterized. From these insights, it is possible to envision the wise use therapies combining standard agents with novel ones that target metabolism to eliminate different subpopulations in tumor tissue, in contrast to therapies which presume tumor cancer cells to be monoclonal. Tumor heterogeneity may affect response to specific drug therapies. Thus, there is a need to provide a means to distinguish when cells in a tumor begin to diverge in molecular identity.

Example 2. Studies identify cycling and hypoxic tumor subpopulations.

Studies have illustrated the importance of the heterogeneity of cells in the tumor microenvironment due to pervasive hypoxia that does not occur in normal tissues (Figure PI). The challenge in studying the tumor microenvironment with specific reference to hypoxia and cell proliferation has been limited by tools that allow for the recovery of cells from tumors with specific characteristics for molecular and cellular studies. To date, studies can be performed in situ with fixed tissues subject to histological sections and staining with antibodies. Even with this limitation, insights have been gained. For example, preliminary studies have documented that a Myc-inducible human Burkitt lymphoma model displayed significant heterogeneity in situ when the tumor tissue section was stained for hypoxic regions using pimonidazole (Figures 3A-3C) 39. Pimonidazole, which is injected

intravenously prior to retrieval of the tissue of interest, is a reactive chemical that is activated by reduction in hypoxia. The activated pimonidazole in turn covalently attaches to protein sulfhydryl groups that can then be detected with an antibody that recognizes pimonidazole moiety covalently linked to modified proteins. Hypoxic regions in the B lymphoma P493 system have been studied, and it has been found that regions of the tumor distal to a blood vessel tend to be hypoxic (Figures 1A-1C and Figures 3A-3C). Furthermore, in tumors concurrently labeled with pimonidazole and bromodeoxyuridine (BrdU) incorporation, BrdU exclusive regions were found, as well as exclusively hypoxic regions devoid of proliferating cells with a pulse of BrdU. There are some regions with a mixture of BrdU labeled cells among those that are hypoxic (Figure 3B). These observations suggest that there are cells that are cycling non-hypoxic cells, hypoxic non-cycling cells and hypoxic cycling cells. The regulation of cell proliferation by hypoxia, and the ability of cells to bypass these hypoxic checkpoints have been the subject of previous studies 22,23. While these current studies underscored the heterogeneity within a tumor bed, retrieval and separation of subpopulations of these cells for genomic and biochemical studies was not able to be performed without taking the entire population of tumor cells from the xenograft.

Bulk tumor cells from P493 lymphoma xenografts have been retrieved and preliminary gene expression arrays performed from the xenograft tissue. A hypoxic signature in cells retrieved from xenografts was found, such as the increased expression of the histone demethylase JMJD1A gene, which is a target of HIF-1 40. However, the increased expression was at best an average over the entire population. Although there was a 3-fold induction of JMJD1A mRNA in tumors by microarray analysis, this is likely highly underestimated due to the dilution of the hypoxic xenograft tumor cells with non-hypoxic cells. In fact, JMJD1A is induced ~6-fold in hypoxic P493 cells in vitro. As such, experimental noise by the inability to distinguish and isolate a subpopulation in tumor tissue may mask important genomic changes that are critical for an understanding of the tumor microenvironment. Because in vivo hypoxic cells may behave differently from cells rendered hypoxic in vivo, due to in situ features such as the metabolic interactions between hypoxic and nearby non-hypoxic cells, it is critical to have tools to identify cells that are hypoxic and/or cycling.

Previous methods to evaluate tumor heterogeneity, suffer from the inability to isolate heterogeneous cells for molecular studies (Pujol et al., "Phenotypic heterogeneity studied by immunohistochemistry and aneuploidy in non-small cell lung cancers." Cancer Res. 1989 May 15;49(10):2797-802; Wang et al., "Estimation of tumor heterogeneity using CGH array data." BMC Bioinformatics. 2009 Jan 9;10:12; Hu et al., "Ultrasensitive, multiplexed detection of cancer biomarkers directly in serum by using a quantum dot-based microfluidic protein chip." ACS Nano. 2010 Jan 26;4(l):488-94). Visual probes using a reporter construct having dual functions (cell cycle and hypoxia probes) would be a powerful reporter for studying tumor heterogeneity. The existence of tumor cell heterogeneity in the tumor microenvironment along with findings regarding metabolic reprogramming by MYC and HIF-1 will lead to the answer of questions, including how the tumor microenvironment reprograms the genomic network of cancer cells; how the tumor microenvironment affects cell growth and proliferation mediated by MYC; how the tumor microenvironment affects key metabolic nodal points; and how inhibitors of key metabolic nodal points affect subpopulations of cancer cells in a tumor. Example 3. A dual reporter construct identifies heterogeneous tumor subpopulations having cycling and hypoxic cells.

The present invention is directed to an innovative tool that marks tumor cells such that cells which are cycling are labeled with mCherry (red) and those that are hypoxic are labeled with green fluorescence protein (eGFP) or a flavin mononucleotide-based fluorescent protein (FbFP; evoglow^®) (Figure 4). This system has been termed HypoxCR. A vector termed HypojcCR-egfp-mcherry was constructed in a lentiviral vector backbone with the reporters arranged bidirectionally and having a puromycin selectable marker (Figure 4). In this vector, the cell cycling reporter comprises a fusion of the fluorescent protein mCherry with 110 amino acids of geminin, a protein that is selectively degraded in the Gl -phase of the cell cycle. This fusion protein was stable in the S/G2M phases of the cell cycle and hence marked cells that were cycling and dividing with mCherry (Figure S2). The synthetic geminin cDNA sequence was designed with optimized human codons. In ΗΕΚ 293T expressing the

HypojcCR-egfp-mcherry vector imaged immediately after 24 hours of 2% oxygen, green hypoxic cells with red cycling cells scattered (Figure 4). Occasional hypoxic cycling

(yellow) cells were observed (Figure 4).

A fluorescent reporter protein was constructed that would be induced by hypoxia through a hypoxia-responsive element and that would have a sufficiently short half-life to reflect real-time hypoxia (Figure 5). Such a probe is not only important for the retrieval of hypoxic cells from tumors for ex vivo studies, but this probe can also be used for in situ imaging of hypoxic tumor regions. For the hypoxic fluorescent reporter, the hypoxic responsive element (HREx5; five copies) was chosen driving fluorescent reporters either by themselves or fused to the oxygen dependent domain (ODD) of HIF-1 a. Under hypoxia, HIF-1 levels increase and HIF-1 binding trigger transactivation of the HREx5. Although it was expected that the fusion comprising the ODD (HIF-1 a residues 548-603) would confer a sufficiently short half-life when cells were no longer hypoxic, it was found that GFP or ODD- GFP or ODD-dsRed (the ODD^548"603 constructs were gifts of Hiroshi Harada⁴¹) all had long half-lives of more than 48 hours and hence were not suitable for real-time reporting of cellular hypoxia. Further fusions with ODD-GFP-PEST and ODD-mRFP-PEST were constructed having a longer fragment of ODD of HIF-la (PCR generated ODD sequence encoding amino acid residues 530-603 42 ) driven by HREx5 (Figure 6). These reporters had properties desirable for study in vivo (Figure 6). These observations indicated that these reporters are feasible and have features that are suitable to probe hypoxia in the tumor microenvironment.

Based on these studies with the hypoxia reporter, the HypojcCR-egfp-mcherry vector was constructed comprising a hypoxia reporter having five hypoxia response elements (HREs) driving a cDNA encoding the fusion of the HIF-1 oxygen dependent domain (ODD) with eGFP and the ornithine decarboxylase (ODC) PEST sequence (Figure 4). This reporter was designed to provide a robust hypoxic response, but with a fluorescent protein that has a half-life which is shorter than one cell cycle time. In this case, in vitro studies revealed that the hypoxia-induced eGFP fusion protein lasted less than 8 hours in normoxic conditions, with the ODD and PEST sequences being critical for keeping the half-life (~4 h) short. In contrast, eGFP fluorescence was observed well after 48 hours of re-oxygenation without the ODD and PEST sequences.

The second system incorporated into the HypojcCR-egfp-mcherry vector construct was a fluorescent reporter protein that marks cycling cells. A system has been developed by exploiting the inherent proteosomal system that is responsible for the degradation of cell cycle protein in specific phases of the cell cycle 43. The fusion of a fluorescent protein with the Ctdl protein allows for the stabilization of the fusion protein in the Gl phase of the cell cycle, whereas fusion with the Geminin protein permits stabilization in the S and G₂-M phases of the cell cycle. Geminin is absent during Gl and accumulates through S, G2, and M phases.

The Geminin fusion proteins were chosen to mark G₂-M cells that are cycling, as has been observed with stably transfected Hela and 293T cells (Figure 6). To track cycling cells, stably transfected Hela and 293T cells were generated with the green fluorescent reporter

GFP fused to Geminin (GFP-Gem). These transfected cells were most fluorescent when they were in the S-G2M phases of the cell cycle (Figure 6). A synthetic Flag-tagged Geminin fused to the green fluorescent protein hmAGl (Flag-hmAGl-Gem) has also been constructed and this construct behaved virtually identically to the original version (Figure 6, middle panel), except protein levels could be detected with anti-Flag tag antibody (not shown). Furthermore, stable 293T cell lines have been generated transfected with both the Flag- hmAGl-Gem and the 5xHRE-ODD-mRFP-PEST to characterize the behavior of these reporters that in combination allow for the determination of hypoxia and cell cycling in single cells (termed HypoxCR system). Using HypoxCR in 293T cells, cell monolayers only displayed green (cycling S-G2M phase cells) (Figure 8C), while transformed foci with thousands of cells piled on top of one another display green or red fluorescence, which is only evident within the foci (Figure 9A). This observation indicates that there is local hypoxia within a transformed focus.

The HypoxCR vectors have been refined to incorporate an insulator sequence between the two promoters of the dual reporter system (Figures 10A and 10B). The insulator prevents crosstalk between promoters (Hasegawa et al., FEBS Lett. 2002 Jun 5;520(l-3):47-52) (Figure 11). Attempts to reduce crosstalk by arranging the transcription of the reporters in the same direction were unsuccessful, possibly because of interference with the polyA tail or the presence of G-quadruplexes (Figure 12). Crosstalk is a consequence of promoters having bidirectional promoters in proximity to each other such that promoters are simultaneously active. For example, in studies using two-color flow cytometry four distinct sets of cells have been identified: hypoxic, non-cycling; hypoxic, cycling; non-hypoxic, non-cycling; non- hypoxic, cycling (Figure 9B). Insulators also decrease or prevent the influence of other nearby DNA sequences, including other promoters and enhancers, on transcription of the reporter.

The use of insulator sequences reduced experimental noise to enhance contrast between cycling and hypoxic cells. Two constructs were created in which the insulator was positioned at one end of promoters transcribing in the same direction (plvx-THRE-mcherry- Geminin) or between two bidirectional promoters (plvx-HRE-I-mcherry-Geminin) (Figures 13A and 13B). At 4 hrs after hypoxia 293T cell transfected with either construct GFP expression was most visible, coinciding with 4 hrs required for reoxygenation of the GFP chromophore. The cells transfected with the plvx-I-HRE-mcherry-Geminin construct showed cells having expression of both eGFP and mcherry at 4hrs (Figure 14). In contrast, cells transfected with the plvx-THRE-mcherry-Geminin construct showed cells having distinct patterns of expression of either eGFP or mcherry at 4hrs (Figure 15). Flow sorting of the cell transfected with each of the constructs also produced similar observations. Cells transfected with the plvx-I-HRE-mcherry-Geminin construct were constitutively green after exposure to hypoxia due to promoter crossstalk. Thus, overlapping peaks of normoxic and hypoxic cells were observed when analyzed by FACS analysis (Figure 16, right). In contrast, cells transfected with the plvx-I-HRE-mcherry-Geminin construct showed distinguishable peaks of normoxic and hypoxic cells by FACS analysis (Figure 16, left). Thus, having an insulator between the two promoters was able to distinguish between normoxic and hypoxic cells. Using the plvx-HRE-I-mcherry Geminin construct, transfected cells displaying heterogeneity can be cultured and expanded for further study, in contrast to other methods of assessing heterogeneity (Figures 17 and 18).

The use of an oxygen independent fluorescence protein (evoglow) increased sensitivity of detection of hypoxia. In contrast to GFP, evoglow fluoresces under hypoxic conditions (Drepper et al., "Reporter proteins for in vivo fluorescence without oxygen." Nat Biotechnol. 2007 Apr;25(4):443-5). At 0 hrs after hypoxic treatment, evoglow expression was highly detectable, but had diminished almost to background levels by 4 hrs. Thus, evoglow increased the sensitivity of the dual reporter and shortened the time for the reporter to identify hypoxic cells.

Figure 21 is a summary of reporter plasmids and their properties.

Example 4. Heterogeneous tumor subpopulations having cycling and hypoxic cells can be studied using the dual reporter.

The heterogeneously marked cells will serve as initial models for studies in vitro under hypoxic and non-hypoxic conditions. Xenografts formed in SCID or nude mice with these cells will be used to pilot in situ imaging studies (e.g., 2-photon fluorescent microscope with which in vivo tracking) as well as retrieval of the xenograft cells for flow cytometry. Based on these studies, a combined single lentiviral construct with both markers would be a very useful tool for tumor biology.

For proof-of-principle, HEK 293T cells, which were easily infected with viral particles containing the HypoxCR reporter, for study in vitro and in situ as xenografts.

Results from xenografts of HEK 293T cells infected with viral particles containing the HypoxCR reporter demonstrated the successful use of the HypoxCR double reporter system in a xenograft model in vivo. The drug- selected HEK 293T cells formed tumors 3 weeks after injection of ~5 x 10⁶ subcutaneously into SCID mice. A tumor-bearing mouse was transcardiac perfused with PBS followed by a 4% formaldehyde solution. The tumor was resected, cut at 2mm from the skin surface and mounted with Vetbond glue to a small petri dish. The tumor was bathed in saline and imaged in situ with a Zeiss LSM510META confocal with a Coherent Chameleon 2-photon laser tuned to 750 nm for mCherry and 910 nm for GFP. A 20x 1.0 NA water dipping objective was used to acquire image stacks of both channels (two separate acquisitions) at a z-step of 3 mm. The images were reconstructed using Bitplane Imaris 3-dimensional image analysis software.

A 215 mm stack was acquired from the cut side of the tumor, which was 2 mm into the tumor (x and y dimensions were 450 mm). The images revealed clusters of hypoxic 293T tumor cells (green) from several angles of the reconstructed 3-D image (Figures 22B-22D). Cycling cells (red) were grouped around clusters and cords of hypoxic cells (green) with occasional hypoxic and cycling cells observed (yellow) (Figures 22B-22D). Example 5. Drug screening of compounds targeting hypoxic cells.

To determine how the tumor microenvironment sensitizes tumor cells to the effects of small molecule inhibitors of glycolysis (lactate dehydrogenase A inhibitor FX11 and 2- deoxyglucose) or of mitochondrial function - FK866 (NAMPT inhibitor of NAD synthesis) and BPTES (glutaminase inhibitor). The HypoxCR system was used to determine which subpopulations of tumor cells in the in situ microenvironment respond to agents that target tumor metabolism and to selected standard chemotherapeutic agents (Figurea 23A-23D).

Without being bound to a particular theory LDHA (which targets glycolysis) would affect a different subset of tumor cells in vivo as compared with FK866, which as an NAD synthesis inhibitor affects mitochondrial function. In particular, hypoxic cells would be susceptible to the LDHA inhibitor, while FK866 or BPTES would affect cells in the central cuff of cells surrounding the tumor blood vessel.

Fluorescent HypoxCR reporters were used to determine the sensitivity of

subpopulations of tumor cells to therapy in vivo and establish complementation between drug-like molecules that target different tumor cell subpopulations.

The LDHA inhibitor (FXl 1) has been shown to have significant in vivo effects in a human lymphoma model and on pancreatic cancer tumor xenografts. BPTES and its derivatives were also found to inhibit glutaminase and are effective against P493 lymphoma xenografts in vivo. FK866, an inhibitor of NAMPT involved in NAD synthesis, had effects on lymphomagenesis such that the combination of FK866 and FXl 1 caused tumor regression. Without being bound to a particular theory, the synergy between FK866 and FXl 1 could either result from their synergistic effect equally affecting all subfractions of a tumor or the from the differential killing of different subsets of tumor cells within the tumor bed. Using the dual reporter approach to studying metabolic targeting for therapeutics will be valuable for future studies of combination therapies with currently available standard of care cancer drugs. Specifically, comparison studies of HypoxCR fluorescently labeled (with 5XHRE- ODD-RFP-PEST and hGEM-hmAGl) P493 lymphoma xenografts that are untreated or treated are performed after they reach an initial size of 300 mm . Tumor xenograft cells are retrieved by flow cytometry to determine if the fraction of cells is different after treatment.

FX11 decreased the population of hypoxic cells relative to control vehicle (DMSO) treated or FK866 treated animals (Figure 24). In the case of FK866 treated tumors, a relative increase in hypoxic cells from the tumors and a decreased pool of cycling cells is expected. BPTES, which is a glutaminase inhibitor, is expected to diminish non-hypoxic cycling tumor cells (both green and red fluorescence), although it is possible that glutaminolysis may continue in hypoxic cells and hence, some decrease in hypoxic cells could also occur. Other inhibitors shown in Figure 24, such as DCA, AOA, and EGCG, as well as 3-bromopyruvate (although not a specific glycolytic inhibitor) are used to assess the response of tumor subpopulations. Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

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Claims

What is claimed is:

1. An isolated nucleic acid molecule comprising a bidirectional reporter cassette that encodes two reporters comprising a first reporter polypeptide fused to a destabilizing moiety, wherein the first reporter selectively identifies a cycling cell and a second reporter polypeptide fused to a destabilizing moiety, wherein the second reporter selectively identifies a hypoxic cell, wherein the expression of the second reporter polypeptide is under the control of a regulatory element that is selectively expressed under hypoxic conditions.

2. The isolated nucleic acid molecule of claim 1, wherein the first and second reporter is selected from the group consisting of GFP, RFP, BFP, CFP, YFP, mCherry, and EvoGlow.

3. The isolated nucleic acid molecule of claim 1, wherein the destabilizing moiety is selected from the group consisting of PEST domain, geminin motif, or fragments or analogs thereof.

4. The isolated nucleic acid of claim 1, wherein the regulatory element is a hypoxic responsive element, an oxygen dependent domain (ODD) of HIF-Ι , or fragments or analogs thereof.

5. The isolated nucleic acid molecule of claim 1, wherein the ODD is HIF-Ια residues 548-603.

6. The isolated nucleic acid molecule of claim 1, wherein the first reporter fused to the destabilizing moiety is separated from the regulatory elements directing expression of the second reporter by an insulating sequence.

7. The isolated nucleic acid molecule of claim 1, wherein the insulating polynucleotide sequence is about 1500 kb in length.

8. The isolated nucleic acid molecule of claim 1, wherein the first and second reporter polypeptides have a half-life that is about equal to the time required for cell division.

9. The isolated nucleic acid molecule of claim 1, wherein the first reporter polypeptide has a half life that is about 5, 10, 15, 20, 24, or 26 hours.

10. The isolated nucleic acid molecule of claim 1, wherein the second reporter polypeptide has a half life that is about 3, 4, 5, 6, 7, 8, 9, or 10 hours.

11. The isolated nucleic acid molecule of claim 1, wherein the first reporter polypeptide is under the control of a promoter that is selectively expressed in dividing cells.

12. The isolated nucleic acid molecule of claim 1, wherein the promoter is the CMV promoter, beta-actin promoter, SV40 promoter-enhancer, or phosphoglycerate kinase (PGK) promoter.

13. The isolated nucleic acid molecule of claim 1, wherein the promoter controlling expression of the first reporter and the regulatory element controlling expression of the second reporter are separated from surrounding nucleic acid sequences by an insulating polynucleotide sequence.

14. The isolated nucleic acid molecule of claim 1, wherein each of the two bidirectional reporters is linked to a selectable marker.

15. The isolated nucleic acid molecule of claim 14, wherein the selectable marker is puromycin, hygromycin, or neomycin.

16. The isolated nucleic acid molecule of claim 1, wherein each of the two reporters is detectable by fluorescence.

17. The isolated nucleic acid molecule of claim 16, wherein the fluorescence emitted by the reporters is at distinct and distinguishable wave lengths.

18. The isolated nucleic acid molecule of claim 1, wherein the first reporter is GFP and the second reporter is mCherry.

A vector comprising the isolated nucleic acid molecule of any of claims 1-18.

20. The vector of claim 19, wherein the vector is an expression vector suitable for expression in a mammalian cell.

21. The vector of claim 20, wherein the expression vector is a viral or non-viral expression vector.

22. The vector of claim 19, wherein the viral expression vector is derived from a lentivirus, adenovirus, retrovirus, adeno-associated virus, herpesvirus, vaccinia virus or polyoma virus.

23. An expression vector comprising a bidirectional reporter cassette that encodes a first reporter comprising a HIF-1 oxygen dependent domain (ODD) fused to a GFP polypeptide fused to a PEST moiety and a second reporter comprising an mCherry polypeptide fused to a geminin polypeptide, wherein the expression of the second reporter polypeptide is under the control of an HRE regulatory element and wherein an insulator sequence is located between the two reporters.

24. An expression vector comprising a bidirectional reporter cassette that encodes a first reporter comprising a HIF-1 oxygen dependent domain (ODD) fused to an evoglow polypeptide fused to a PEST moiety and a second reporter comprising an mCherry polypeptide fused to a geminin polypeptide, wherein the expression of the second reporter polypeptide is under the control of an HRE regulatory element and wherein an insulator sequence is located between the two reporters.

25. An expression vector comprising a bidirectional reporter cassette that encodes a first reporter comprising a HIF-1 oxygen dependent domain (ODD) fused to a GFP polypeptide fused to a PEST moiety and a second reporter comprising an mCherry polypeptide fused to a geminin polypeptide, wherein the expression of the second reporter polypeptide is under the control of an HRE regulatory element, wherein an insulator sequence is located between the two reporters, and wherein the bidirectional reporter cassette is located between two insulator sequences.

26. An expression vector comprising a bidirectional reporter cassette that encodes a first reporter comprising a HIF-1 oxygen dependent domain (ODD) fused to an evoglow polypeptide fused to a PEST moiety and a second reporter comprising an mCherry polypeptide fused to a geminin polypeptide, wherein the expression of the second reporter polypeptide is under the control of an HRE regulatory element and wherein an insulator sequence is located between the two reporters, and wherein the bidirectional reporter cassette is located between two insulator sequences.

27. An expression vector comprising a reporter cassette that encodes a first reporter comprising a HIF-1 oxygen dependent domain (ODD) fused to a GFP polypeptide fused to a PEST moiety and a second reporter comprising an mCherry polypeptide fused to a geminin polypeptide, wherein the expression of the second reporter polypeptide is under the control of an HRE regulatory element, wherein the first reporter is located upstream of the second reporter, and wherein an insulator sequence is located upstream of the first reporter.

28. A host cell comprising the vector of any one of claims 14-18.

29. The host cell of claim 28, wherein the cell is in vitro, in vivo, or ex vivo..

30. The host cell of claim 28, wherein the cell is a mammalian cell.

31. The host cell of claim 30, wherein the cell is a human cell.

32. The host cell of claim 28, wherein the cell is derived from a tumor or immortalized cell line.

33. The host cell of claim 28, wherein the cell is a HeLa cell, 293T cell, P493 lymphoma cell, or a P198 cell.

34. A xenograft comprising the cell of any of claims 28-33.

35. A transgenic non-human animal comprising an expression vector comprising a bidirectional reporter cassette that encodes a first reporter polypeptide fused to a destabilizing moiety, wherein the first reporter selectively identifies a cycling cell and a second reporter polypeptide fused to a destabilizing moiety, wherein the second reporter selectively identifies a hypoxic cell, wherein the expression of the second reporter polypeptide is under the control of a regulatory element that is selectively expressed under hypoxic conditions.

36. The transgenic animal of claim 35, wherein the animal is a mammal.

37. The transgenic animal of claim 36, wherein the mammal is a rodent.

38. The transgenic animal of claim 37, wherein the rodent is a mouse or rat.

39. A method for classifying tumor cells as hypoxic cells, cycling cells, or cycling hypoxic cells, the method comprising

(a) expressing in the cells an expression vector of any of claims 14-19; and

(b) detecting the expression of the first and second reporters in said cells.

40. The method of claim 39, further comprising characterizing the expression of a polypeptide selected from the group consisting of HIF, MYC, HK2, PKM2, LDHA, PDK1, MCT1, GLUD1 (glutamate dehydrogenase), and GPT (glutamate pyruvate transaminase) or the polynucleotides encoding them.

41. A method for isolating one or more tumor cell subpopulations, each cell

subpopulation comprising hypoxic, cycling, or cycling hypoxic cells, the method comprising

(a) expressing in the cells an expression vector of any of claims 14-19;

(b) detecting the expression of the first and second reporters in said cells; and

(c) isolating a population of cells enriched for expression of the first reporter, isolating a subpopulation of cells enriched for expression of the second reporter, and/or isolating a population of cells enriched for expression of the first and second reporters, wherein each of said cell subpopulations are enriched for hypoxic, cycling, or cycling hypoxic cells, respectively.

42. The method of claim 41, wherein the cells are isolated using fluorescence activated cell sorting (FACS) or Laser-Enabled Analysis and Processing (LEAP) microplate -based cytometry.

43. The method of claim 41 or 42, wherein said first and second reporters are fluorescent proteins that emit at distinct and distinguishable wave lengths.

44. A method for characterizing the chemotherapuetic activity of an agent, the method comprising

(a) expressing in a population of cells an expression vector of any of claims 14-19, wherein the population comprises cells exposed to normoxic and hypoxic conditions;

(b) contacting the cells with a chemotherapeutic agent;

(c) detecting an alteration in the survival or the proliferation of the cells;

(d) detecting the expression of the first and second reporters in any surviving cells, wherein the disproportionate survival of a cell expressing a hypoxic reporter characterizes the chemotherapeutic agent as ineffective in reducing the survival or proliferation of hypoxic cells, and the disproportionate survival of cycling cells characterizes the chemotherapeutic agent as ineffective in reducing the survival or proliferation of cycling cells.

45. A method for identifying an agent that reduces the proliferation or survival of a hypoxic cell that is refractory to conventional chemotherapy, the method comprising

(a) expressing in a population of cells an expression vector of any of claims 14-19, wherein the population comprises cells exposed to normoxic and hypoxic conditions;

(b) contacting the cells with a chemotherapeutic agent and detecting a reduction in the survival or the proliferation of the cells;

(c) detecting the expression of a hypoxic reporter in the surviving cells, and

(d) exposing the surviving cells of step (c) to a second agent, and detecting a reduction in the survival or proliferation of the surviving cells of step (c), thereby identifying the agent as reducing the proliferation or survival of a hypoxic cell that is refractory to conventional chemotherapy.

46. The method of claim 45, wherein the chemotherapeutic agent is hydroxyurea or another agent that inhibits ribonucleotide reductase or otherwise inhibits cell cycling.

47. The method of claim 45, wherein the chemotherapeutic agent is gemcitabine.

48. The method of claim 45, wherein the second agent is Cytoxan or an LDHA inhibitor.

49. The method of claim 45, wherein the cell is derived from a tumor or immortalized cell line.

50. The method of claim 45, wherein the cell is a HeLa cell, 293T cell, P493 lymphoma cell, or a P198 cell.