Methods for identifying stem cells and other cells specific to embryogenesis and carcinogenesis, classifying tissue samples, diagnosing precancerous and cancerous or atherosclerotic lesions, testing the value of anticancer agents, discovering macromolecules specifically expressed in particular cell types, using stem cells in restorative tissue therapy as well as methods for preparing tissue samples so heteromorphic nuclear morphotypes remain intact are disclosed.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention derives from the process of identification of and method for classifying organ-specific and/or tumor stem cells and other specific cell forms in a cell culture, tissue, pre-neoplastic lesion or tumor sample. The invention relates to the process of determining the nuclear morphotypes, the modes of nuclear division and the involvement of nuclei of particular morphotype in multicellular aggregates and multinuclear syncytia among all cells in a cell culture, tissue, preneoplastic lesion or tumor sample and identification of organ-specific or tumor stem cells on the basis of a particular nuclear morphotype alone. Multiple forms (nuclear morphotypes) of clear and reproducible non-spherical nuclei in fetal tissue, preneoplastic lesions (adenomas of the colon) and neoplastic lesions or tumors (adenocarcinomas of the colon, carcinomas of the pancreas) were observed that were absent in normal adult tissue such as colonic crypts or liver parenchyma. These morphotypes, disclosed herein, included nuclei of size and shapes previously unreported in the annals of histology or pathology of human tissues.

The nuclear forms or morphotypes in fetal and neoplastic tissues included the spherical and ovoid nuclear forms commonly seen in adult tissues but also presented a diverse set of reproducible morphotypes that ranged in size from the 40 micron "sausage-shaped", through shorter (.about.8-20 micron) "cigar-shaped", "bullet-shaped" and "kidney shaped" to "condensed spherical" nuclei of some 4 microns diameter. One remarkable, previously unreported, nuclear morphotype had the form of cups or bells with an open "mouth''" designated "bell-shaped". These bell-shaped nuclei were observed in symmetric nuclear divisions resembling the separation of two stacked paper cups.

These "cup-from-cup" divisions were also remarkable in that they were amitotic, e.g., did not involve condensation of all human chromosomes and separation as in mitosis. They were symmetrical nuclear divisions insofar as two apparently identical bell-shaped nuclei resulted from the cup-from-cup division. These bell-shaped nuclei were also observed to undergo amitotic asymmetric nuclear divisions in which a bell-shaped nucleus appeared to give rise to an enclosed nucleus of one of the other nuclear morphotypes. This production of the original bell-shaped nucleus and a nucleus of a different morphotype is the first known visualization of an asymmetrical nuclear division. The appearance of a heteromorphic nuclear morphotype distinct from the bell-shaped nucleus in asymmetrical amitotic cell divisions involving bell-shaped nuclei is disclosed herein to distinctly define a cell with a heteromorphic nuclear morphotype distinct from the phenotype of a cell with a bell-shaped nucleus.

Asymmetric nuclear division is widely considered as a necessary characteristic of stem cells in normal development. The discovery of nuclear morphotypes common to both fetal tissue, preneoplasia and neoplasia bear on the hypothesis that tumors are a re-expression of embryonic phenotypes, specifically stem cells forming clonal populations with derived differentiated cellular phenotypes. The nuclear morphotype that identifies a stem cell appears as a bell- or cup-shaped nucleus in which stained DNA creates a hollow structure easily differentiated from all other nuclear morphotypes in a fetal tissue or tumor sample in which stained DNA images show a nucleus fully encased by a DNA-containing structure.

Further observations confirmed and extended the discovery that adenocarcinomas and embryos partially share lineages of cells with distinct nuclear morphotypes arising from what appears to be the identical processes of symmetrical and asymmetrical nuclear divisions of bell-shaped nuclei without the appearance of a mitotic apparatus. While the picture of the appearance and disappearance of these nuclear forms from early embryo through fetal and juvenile growth to adult organ and reappearance in carcinogenesis is incomplete, the potential value of these findings in cancer prevention and therapy is of obvious importance offering benefits in diagnosis and treatment of cancers and other diseases such as atherosclerosis that also arise from slowly growing monoclonal colonies. Benefits are expected from growing these bell-shaped nuclei independently for applications such as, for example, restorative therapy for complex tissues and organs.

The value of the process described herein to specifically identify these previously unrecognized cells as stem cells by their nuclear morphotype, modes of nuclear division and/or involvement in multi-nuclear structures in normal and tumor tissues is clear. The claimed processes make it possible to specifically recognize stem cells and other fetal and tumor-specific nuclear morphotypes, permit their isolation and study, and provide for their use in tests to discover which of many plausible preventative or therapeutic regimens for cancer are effective. The methods described herein also provide means to discover specific stem cells for regeneration and transplantation therapies for human tissues and organs (e.g., tissue restoration therapy).

In one embodiment, the invention is directed to a method for characterizing (e.g., classifying) a cell or tissue sample based on nuclear structures associated with stages of development or pathology, comprising: a) visualizing the nuclei of cells distributed throughout the tissue sample, wherein the tissue sample is prepared by a method that substantially preserves the integrity of structures of substantially all nuclei having maximum diameters up to about 50 microns; and b) determining the presence and/or absence of a class or classes of nuclear morphotypes, wherein presence or absence of a particular class is indicative of a stage of development or pathology. In one embodiment, the tissue sample is obtained by surgical excision. In a particular embodiment, the tissue sample is physically (e.g., frozen) or chemically fixed (e.g., treated with one or more chemical fixing agents selected from the group consisting of: alcohols, aldehydes, organic acids and combinations thereof such as, for example, methanol and acetic acid). In a particular embodiment, the tissue sample is fixed prior to cellular degradation of nuclei. In another embodiment, the cells of the tissue sample are partially dissociated by tissue maceration and spreading. In one embodiment, the cells or macromolecules of the tissue sample are stained, thereby allowing visualization of nuclei. In another embodiment, DNA is stained, thereby allowing visualization of nuclei. In another embodiment, the tissue sample is fixed within 30 minutes of surgical removal.

In another embodiment, the tissue sample is obtained from a multicellular animal (e.g., a vertebrate, e.g., a mammal, e.g., primates, rodents, canines, felines, porcines, ovines, bovines and rabbits). In a particular embodiment, the mammal is a human.

In a particular embodiment, the presence or absence of a particular class or classes of nuclear morphotypes is indicative of a particular stage of development (e.g., embryonic, fetal (organogenesis), neonatal, juvenile and adult stages of development). In another embodiment, the presence or absence of a particular class or classes of nuclear morphotypes is indicative of a tissue sample selected from the group consisting of: normal, preneoplastic, neoplastic and metastatic. The class or classes of nuclear morphotypes is selected from the group consisting of: bell-shaped, cigar-shaped, condensed spherical, spherical, oval, sausage-shaped, kidney-shaped and bullet-shaped.

In another embodiment, the methods of the present invention further comprise determining the spatial or numerical distribution of one or more classes of nuclear morphotypes within the tissue sample, wherein the spatial or numerical distributions of the one or more classes of nuclear morphotypes further characterizes the tissue sample. A particular spatial or numerical distribution is indicative of a normal, preneoplastic, neoplastic or metastatic tissue.

In another embodiment of the invention, the nuclei are contained in multinuclear syncytia or in mononuclear cells and wherein the tissue sample is adult tissue. In one embodiment, the presence of bell-shaped, cigar-shaped or bullet-shaped nuclei are indicative of preneoplasia, neoplasia or metastasis. In another embodiment, the appearance of multinuclear syncytia is indicative of neoplasia or metastasis. In one embodiment, structures indicative of amitotic symmetrical nuclear division are indicative of neoplasia or metastasis. In another embodiment, the presence of bell-shaped nuclei and the absence of multinuclear syncytia are indicative of preneoplasia. In yet another embodiment, the presence of non-spherical and non-oval nuclei in blood vessel wall tissue is indicative of an incipient atherosclerotic condition.

In another embodiment, the invention is directed to a method for identifying a cell of interest or multinuclear syncytium of interest in a cell culture or tissue sample, wherein the cell of interest or syncytium of interest is identified by visualizing nuclear morphology, wherein the cell of interest comprises a heteromorphic nuclear morphotype. In a particular embodiment, the cell or multinuclear syncytium is isolated from the tissue sample. In another embodiment, the cell of or multinuclear syncytium is isolated by microdissection, e.g., pressure-assisted laser microdissection. In one embodiment, the cell of interest or multinuclear syncytium is identified in a population of cells in culture. In another embodiment, the cell of interest or multinuclear syncytium is identified in a tissue sample. In another embodiment, the cell of interest or syncytium of interest comprises one or more amitotic nuclear division complexes. In yet another embodiment, the cell of interest is present within a multicellular aggregate of cells. In a particular embodiment, the cell of interest is present within a cluster of multicellular aggregates.

In another embodiment, the invention is directed to a cell of interest isolated or identified by the methods disclosed herein. In one embodiment, the invention is directed to a method for using a cell of interest isolated or identified by the methods disclosed herein to identify one or more macromolecular markers specific to the cell of interest, wherein the marker is indicative of a particular stage of development or pathology. In a particular embodiment, the macromolecular marker is an antigen, cell-surface marker, nucleic acid, protein, phosphorylated protein or glycosaminoglycan.

In another embodiment, the invention is directed to a method for diagnosing preneoplasia or neoplasia comprising identification of one or more macromolecular markers, wherein the identification of the one or more macromolecular markers in adult tissue is indicative of preneoplasia or neoplasia.

In another embodiment, the invention is directed to a method of identifying one or more anti-tumorigenic agents comprising: a) treating a mammal having a tumor with one or more candidate agents; b) determining the nuclear morphology of cells contained within a tumor sample obtained from the mammal; and c) comparing the nuclear morphology of the cells from the mammal treated with the candidate anti-tumorigenic agent with cells obtained from a mammal having a tumor but not treated with the candidate anti-tumorigenic agent, wherein elimination of cells comprising neoplastic nuclear morphotypes is indicative of the effectiveness of the agent as an anti-tumorigenic agent. In a particular embodiment, the neoplastic nuclei are selected from the group consisting of: bell-shaped nuclei, cigar-shaped nuclei and bullet-shaped nuclei. In another embodiment, the alteration in nuclear morphology comprises the elimination of bell-shaped nuclei. In one embodiment, the mammal is a rodent (e.g., a rat or mouse. In a particular embodiment, the nuclei are arranged in syncytia and the elimination of neoplastic nuclear morphotypes comprises a disruption of the syncytia.

In another embodiment, the invention is directed to a method of identifying one or more anti-tumorigenic agents comprising treating a cultured tumor tissue or cell sample with one or more candidate agents and evaluating the nuclear morphology of cells contained in the tumor sample, wherein the cells comprise heteromorphic nuclear morphotypes, wherein in the absence of an anti-tumorigenic agent, the cultured tumor cells maintain their heteromorphic nuclear morphotypes, and wherein the elimination of neoplastic nuclear morphotypes is indicative of the effectiveness of the agent as an anti-tumorigenic agent, and wherein the elimination of preneoplastic nuclear morphotypes is indicative of a tumor preventative agent. In a particular embodiment, the altered nuclear morphology comprises the elimination of one or more nuclear morphotypes selected from the group consisting of: bell-shaped nuclei, cigar-shaped nuclei and bullet-shaped nuclei. In one embodiment, the alteration in nuclear morphology comprises the elimination of bell-shaped nuclei. In one embodiment, the nuclei are arranged in syncytia and the elimination of neoplastic nuclear morphotypes comprises a disruption of the syncytia.

In another embodiment, the invention is directed to a method for preparing a mammalian tissue sample suitable for the identification of cells comprising nuclei having maximum diameters up to about 50 microns, comprising: a) disrupting cellular adhesions of cellular sheets of the tissue sample; and b) spreading the cells with disrupted adhesions onto a hard surface, wherein the structural integrity of the nucleus of the cells remains intact, thus rendering the sample suitable for the identification of heteromorphic nuclear morphotype cells. In one embodiment, the tissue sample is sectioned into layers wherein the layers obtained exceed the thickness of a cell. In another embodiment, the tissue sample forms a layer on the microscope slide of about 0.5 millimeters. In a particular embodiment, the tissue sample forms a layer greater than about 50 microns. In another embodiment, cellular adhesions of the tissue sample are chemically disrupted (e.g., by treatment with 45% acetic acid). In a particular embodiment, the fixed, chemically disrupted tissue samples prior to spreading are about 1 mm.sup.2 in area. In a particular embodiment, the tissue sample is a human tissue sample. In a particular embodiment, the cellular sheets are about 1 mm.sup.2 in area. In another embodiment, the hard surface is a microscope slide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the unexpected discovery of large cell nuclei with distinct morphologies throughout fetal gut (5-7 wks), colonic adenomas, and adenocarcinomas, some of which are not present in normal (non-neoplastic) adult colon. These "heteromorphic nuclear morphotypes" (e.g., nuclear morphotypes that differ from the normally spheroid or ovoid nuclei of cells in adult organs), were observed in embryonic tissues and only rarely in adult tissues. One remarkable nuclear morphotype has a nucleus shaped like a hollow bell (approximately 10-15 microns in height and approximately 7-12 microns in bell mouth diameter). These bell-shaped structures appear to divide symmetrically by an amitotic process resembling the separation of two paper cups. Furthermore, at least seven other nuclear forms were observed to emerge from bell-shaped nuclei in asymmetrical amitoses. Cells containing these derivative nuclear forms subsequently divide by mitoses forming clonal populations of identical nuclear morphotypes in embryos, adenomas and adenocarcinomas. Cells with bell-shaped nuclei thus appear to be responsible for both net growth and differentiation in embryonic gut, adenomas and adenocarcinomas and fulfill the requirements for generative, multipotent stem cells in embryogenesis and carcinogenesis. The specific differential occurrence of bell-shaped nuclei in cells demonstrating both symmetrical and asymmetrical amitoses theoretically required for net stem cell growth and differentiation in embryonic/fetal tissue sample as opposed to adult tissue, allows for one skilled in the art of microscopic histology/pathology to classify cells as stem cells or not-stem cells according to their nuclear morphology.

A concept shared in both embryology and oncology is that the multiple cell types that are observed in organs and tumors derived from particular organs arose from precursor "stem" cells capable of net growth by symmetrical cell divisions in which a cell division produces two identical precursor "stem" cells and differentiation by asymmetrical cell division producing one precursor "stem" cell and one differentiated cell. This differentiated cell may then divide an additional number of times to create a large number of differentiated cells that eventually reach a non-dividing terminal stage followed by programmed cell death. An "extinction" division occurs when a cell differentiates into two more highly differentiated cells.

Tumors display many of the characteristics of the adult organ/system from which they are derived including formation of complex multi-cellular structures, such as, for example, colonic crypts in adenocarcinomas of the colon. Insofar as tumors have been derived from a single precursor cell that had the ability of both net growth and differentiation, it has been reasoned that "tumor stem cells" must exist and share many characteristics organ-specific stem cells (partially undifferentiated cells capable of giving rise to specific organ cells and tissues). For example, in the case of the cells of the bone marrow from which leukemias arise, certain antigens have been recognized that allow identification of marrow cell sub-populations that contain one or a small number cells capable of giving rise to a complete blood cell system on transplantation. Similarly a subfraction of tumor cells can be recognized by specific antigens expressed by some tumor cells. According to this logic, one cell among the sub-population so recognized gives rise to a new differentiated tumor upon experimental transplantation of mixtures of tumor cells. Continuing with this reasoning, the "organ-specific stem cells" and "tumor stem cells" exist among such antigen expressing sub-populations. These antigens are used to identify and isolate cell populations containing at least one pluripotent stem cell, but will also identify a large majority of cells that cannot act as stem cells. None of the cells of these populations as isolated have been found to undergo asymmetrical cellular divisions that are considered a "shibboleth" of true stem cells or organs and tumors.

Presented here are methods for identifying cells that are in fact "organ-specific stem cells" or "tumor stem cells" as opposed to subfractions of organs or tumors enriched for such cells. The identification is based on the heteromorphic nuclear morphotypes and nuclear divisions and/or arrangements described herein. With these methods, it would be possible to isolate such stem cells and discover their specific biochemistry and molecular biologies with the goals of isolating cells permitting organ regeneration, interfering with the growth of pre-cancerous lesions and/or the killing of the cells specifically responsible for tumor growth and reappearance after attempts at therapy.

Such organ-specific and tumor stem cells are further identified in fetal organs, tumors and tumor metastases by their participation as a class in two specific previously unreported, microscopically visible forms of nuclear division. In the first of these two forms of nuclear division there is no general condensation of chromosomes and formation of a mitotic apparatus; instead one bell-shaped nucleus appears to give rise to an identical bell-shaped nucleus in a manner similar to separation of two paper cups. This symmetrical, amitotic division provides for the net growth of stem cells in fetal and tumor samples. In the second form of stem cell-specific nuclear division there is also no general condensation of chromosomes and formation of a mitotic apparatus; instead one bell-shaped nucleus appears to give rise to a bell-shaped nucleus and a nucleus of a nucleus having one of the several nuclear morphotypes observed in fetal tissue and tumor samplers (FIG. 3, see Original Patent). This asymmetrical, amitotic division provides for creation of differentiated cells by stem cells in fetal tissue and tumors. Mitotic division of nuclei without bell-shaped nuclei are observed in fetal tissue and tumor samples creating the majority of total cells in such samples. Thus the process of identification of stem cells permits direct observation of cells with bell-shaped nuclei undergoing both symmetrical and asymmetrical nuclear divisions, both necessary for classification as organ-specific and tumor-specific stem cells.

Such organ-specific and tumor stem cells are further identified in fetal organs, tumors and tumor metastases by their participation as a class in specific previously unreported multi-nuclear structures resembling long tubes in which bell-shaped nuclei are regularly aligned in a fashion resembling a series of separated paper cups retaining the head-to-toe relationship as in the stacked cup set.

These stem cell-specific nuclear morphotypes (bell-shaped), specific forms of nuclear division (symmetrical and asymmetrical amitotic nuclear divisions) and specific form of participation in multinuclear structures (long multinuclear tubes) are essentially absent from adult organs. However, the stem cell-specific nuclear morphotype is observed among the cells of preneoplastic lesions and rarely, as single nuclei, widely dispersed in adult organs (for example, somewhat fewer than one in two million nuclei of adult colonic crypts have been found to be bell-shaped nuclei).

The unexpected discovery of these heteromorphic nuclear morphotypes and their differential occurrence in stem cells, normal adult tissue and tumor tissue allows for one skilled in the art to classify cells according to their nuclear morphology. In addition, stem cells can be isolated based on their nuclear morphology, anti-tumorigenic agents can be screened or identified based on the appearance or disappearance of tumor-specific morphotypes, and tissue samples can be classified (e.g., normal or abnormal; neoplastic or non-neoplastic) by determining the morphotypes present in the cells of the tissue. As a result of this cell classification, a diagnosis can be made as to whether or not an individual has cancer or a pre-cancerous lesion. Although a particular method (see below) is used in the Examples to allow for the visualization of these novel morphotypes, any method that allows for the identification and evaluation of nuclear morphotypes is suitable for the classification of cells, tissues and samples, and subsequent diagnosis of disease, as well as provide the basis for assays used to identify anti-tumorigenic agents (e.g., agents effective in inhibiting or decreasing tumorigenic cell growth). Such methods include phase contrast microscopy, confocal microscopy, two electron or two wavelength microscopy and small angle scatter flow cytometry.

For the purposes of the present invention, thick, .about.0.5 mm, sections of normal adult human colonic epithelium, colonic adenomas, colonic adenocarcinomas, and fetal gut were prepared for microscopic observation as described in the Exemplification below. The thickness of the tissue sheet layer should be at least the thickness of an intact cell (and not just a section or slice of the cell). An array of large spheroidal and non-spheroidal nuclear forms appeared in all samples as summarized in FIG. 1 (see Original Patent). All of the samples contained the spheroid and ovoid nuclei normally observed in histological sections of adult colonic crypts but also contained extraordinary, previously unreported nuclear morphotypes. Embryonic tissue contained nuclei shaped like bells, tapered cigars, kidney beans, sausages and small spheres. Normal adult colonic crypts contained an occasional bell-shaped nucleus in the crypt bases but the vast majority were the large spheres and ovoid structures. In some views it appears that cell nuclei near the crypt bases may be "discoid". In adenomas and adenocarcinomas the nuclear shapes, in addition to the spheroid and ovoid nuclei, included tapered cigars and an additional form that looks like a cigar with a bitten off end dubbed "bullet-shaped".

Crypt structures were preserved by the preparative procedure and were clearly observed in normal colon, adenomas and adenocarcinomas. The .about.0.5 mm sample sections employed were much thicker than the largest nuclear form observed, the sausage-shape, which was .about.40 microns in length. All of the nuclear structures, except the 4 micron "condensed spherical nuclei", had at least one internal axis longer than the 5 micron sections usually employed in pathological evaluations. Furthermore, there is a fair degree of morphological variation among nuclei that can be classified as "bell-shaped" or "cigar-shaped" etc., suggesting independent lineages and physiological functionalities.

The phenomena of bell-shaped nuclei, their symmetric and asymmetric forms of amitosis, or the collection of nuclear morphotypes in adult, preneoplastic, neoplastic and embryonic tissue described herein have not been previously reported (see Example 2). The tubular encasement of linearly arrayed bell-shaped nuclei in embryos and adenocarcinomas is also apparently a novel observation. The reason that they have not been previously observed may lie in the differences between standard histological practices and those employed and disclosed herein. Two clear procedural differences are evident. First, all tissues for fixation were sectioned and fixed within a short period of time (for example, within 30 minutes) of surgical removal. Preparations after 30 minutes may begin to show degradation of the nuclear forms, although careful tissue preparation may prevent degradation. Second is the difference between thin section procedures practiced in medical pathology and thick section fixation protocols, disclosed herein- the latter preserving, and the former apparently destroying, the structures/conditions that maintain these nuclear shapes.

Amitosis, as a phenomenon, has been reported in a number of protozoans and primitive metazoans (Orias, E., 1991, J. Protozool., 38:217-221; Prescott, D., 1994, Proc. Natl. Acad. Sci USA, 92:136-140). However, these amitoses were unlike those reported here insofar as protozoan amitotic nuclear division occurred by formation of a nuclear cleft and pinching off two separate approximately equal nuclei (Fujiu, K. and Numata, O., 2000, Cell Motil. Cytoskeleton, 46:17-27). Amitotic divisions of the sort similar to those seen in protozoans have been reported, however, in a number of different tumors (Okuyama, S. 1991, Tohoku J. Exp. Med., 164:247-249; Okuyama, S., 1992, Tohoku J. Exp. Med., 168:445-448; Elias, H. and Fong, B., 1978, Hum. Pathol., 9:679-684; Elias, H. and Hyde, D., 1982, Hum. Pathol., 3:635-639).

The observations showing that the arrangement of chromosomes in early prophase nuclei of the mitotic cells maintains the shape of the interphase nucleus also deserve attention. It appears that the different chromosomes form a highly structured mosaic that may have important consequences in defining a cell's phenotype. The relationship between the spatial arrangement of chromosomes in interphase nuclei and cell physiology is an active area of exploration (Misteli, T., 2001, Science, 291:843-847; Thomas, C. et al., 2001, Proc. Natl. Acad. Sci. USA, 99:1972-1977; Parada, L. et al., 2004, Exp. Cell Res., 296:64-70).

Based on the observations cited below, cells with bell-shaped nuclei are pluripotent cells that represent the generative cell of the developing and growing tumors and preneoplastic lesions such as, for example, colorectal tumors, adenomas and adenocarcinomas. Thus, the bell-shaped nuclear morphotype is indicative of pluripotent stem cells and can be used as diagnostic criteria for preneoplastic and neoplastic tissue in adult tissue samples. In addition, the organization of bell-shaped nuclei (e.g., into tube-like structures or spider-web-like structures) can be further indicative of the progression of tumor development (e.g., neoplasia or metastatic tumors).

The pluripotency of bell-shaped nuclei is demonstrated by the images of multiple forms of nuclei emerging from bell-shaped nuclei in asymmetrical amitotic divisions. Insofar as egg-, sausage, kidney-, bullet- and cigar-shaped nuclei are observed emerging from bell-shaped nuclei and no other nuclear forms are observed, they represent the set of functions necessary for the tissue in which they reside to persist. There can indeed be multiple forms of cells with bell-shaped nuclei as suggested by the morphological variations among bell-shaped nuclei observed.

The numbers and symmetrical amitotic frequencies of cells with bell-shaped nuclei are consistent with the generative element of this hypothesis. They are observed in large numbers in embryos, rare in normal adult colon free of neoplasia, present, in small numbers (for example, about 1,000) in adenomas of a few cubic millimeters and large numbers (for example, less than about 1,000,000) in adenocarcinomas of several cubic centimeters. Their division rates in the embryo and adenocarcinomas are estimated to be approximately 20 divisions per year consistent with the estimated net growth rates of colonic adenocarcinomas (Herrero-Jimenez, P. et al., 1998, Mutat. Res., 400:553-578). Their symmetrical amitotic fraction in adenomas is less than 1/1000 and none have been seen to date. This negative observation is important in itself. The frequency of cell divisions for the generative cell or "cell at risk of promotion" in human colonic preneoplastic lesions has been estimated by calculation to be about one in six years (Herrero-Jimenez, P. et al., 2000, Mutat. Res., 447:73-116). Assuming amitosis could be recognized for a three hour period, a frequency of less than 6/100,000 would be expected. Thus, the very low symmetrical amitotic rate of adenomas is consistent with expectation for the generative cells of colonic preneoplasia.

It is possible that the cells with bell-shaped nuclei are phased out at the end of juvenile growth. Retinoblasts phase into retinocytes in early childhood and remove the risk of retinoblastoma in retinoblastoma gene heterozygotes (Knudson, A., 1971, Proc. Natl. Acad. Sci. USA, 68:820-823). It could be that colon tumor "initiation" by mutations in genes such as APC prevents this phasing out process. If so, bell-shaped nuclei should be found in the bases of colonic crypts in neonates and juveniles.

From the appearance of bell-shaped nuclei in embryonic and carcinogenic tissues, relationships between embryogenesis and carcinogenesis can be inferred. Cancer researchers have considered tumors to reflect characteristics of embryos for more than a century. Erenpresia, J. and Helmtrud, I. (1999, Mech. Aging and Develop., 108:227-238) cited J. Cohnheim (1875, Virchows Arch., 65:64; 1877-1880, Vorelesungen uber allgemeine Pathologie. Ein Handbuch fur Artzte und Studierende. Berlin, Hirchswald 1-2 691S) as first hypothesizing that tumors arise from fetal cells that inappropriately persist in adult tissues. The expression of carcino-embryonic antigens in tumors and appearance in tumors of a wide spectrum of gene products, mRNAs and proteins, that are also found in embryos has reinforced the broad hypothesis that oncogenesis involves the appearance of cells with embryo-like qualities. The finding of morphological cell types essentially identical in form, amitotic and mitotic behavior in adenocarcinomas and embryonic colon calls for a more specific restatement of the carcino-embryonic hypothesis in terms closely echoing Cohnheim and using the more recent arguments and experimental demonstrations indicating the existence of tumor stem cells (Pardal et al., 2003, Nature Rev., 3:895-902).

These new observations, integrated into the body of cancer research and ideas of the past 130 years, suggest a simple hypothesis about the origin and characteristics of late-onset colonic adenomas and adenocarcinomas: tumor initiating mutations, e.g., APC gene inactivations, occur in a cell with a bell-shaped nucleus before this cell form is phased out during or at the end of the juvenile period. Such initiated cells with bell-shaped nuclei would simply continue to divide and create new colonic crypts at the same rate as they did in juveniles (Herrero-Jimenez, P. et al., 1998, Mutat. Res., 400:553-518; Herrero-Jimenez, P. et al., 2000, Mutat. Res., 447:73-116). The resultant local crowding creates the "polyp". Either actively, by an additional genetic change or changes (including changes in gene imprinting), or passively by biochemical changes occurring within the growing adenoma, a single cell with a bell-shaped nucleus reverts to an earlier embryonic condition and gives rise, as in the embryo, to a rapidly growing array of cells almost indistinguishable from embryonic tissue. Untreated, this continued growth leads to colonic obstruction and/or metastases and death.

In the most general sense these observations point to a highly ordered nature of carcinogenesis in which distinctly non-chaotic behavior is observed in adenomas that preserve the slow but constant growth rate of juveniles and in adenocarcinomas that recreate an ordered ensemble of cell types and growth rates observed during embryogenesis. In the sense that the existing biological forms have been selected from a myriad of degenerate possibilities ("trying all combinations"), it is perhaps not surprising that carcinogenesis in humans might represent a rare but simple failure to cease juvenile growth and a subsequent rare reversion to an ordered embryonic cell state.

These observations suggest that cells with bell-shaped nuclei would be targets for more specific and therefore more effective forms of tumor prevention and therapy. Were it possible to drop the net growth rate of preneoplastic colonies by 50%, most late onset cancer types would not appear during a human lifetime of 100 years (Herrero-Jimenez, P. et al., 1998, Mutat. Res., 400:553-578; Herrero-Jimenez, P. et al., 2000, Mutat. Res., 447:73-116). It is reasonable to believe that cells with heteromorphic nuclear morphotypes such as, for example, bell-shaped nuclei are the tumor stem cells in adenomas and adenocarcinomas of the colon, one might target the mechanisms that confer their special characteristics in DNA synthesis and segregation either in symmetrical divisions of net growth or the asymmetric divisions that provide the cells that divide by mitosis and provide the bulk of the tumor mass. It may be that these cell types or entubated bell-shaped nuclei operate under different biochemical rules and that these, if understood, might also be exploited in tumor prevention and/or therapy (see, for example, the findings of Otto Warburg who discovered marked differences in mitochondrial biochemistry among embryonic, adult organ and cancer tissues (Warburg, O., 1956, Science, 123:309-314; Warburg, O. et al., 1960, Z. Naturforsch B., 15B:378-379).

Reference to basic texts on invertebrates shows that a large sausage-shaped nucleus exists among the ciliated protozoans such as the pertirich Vorticella and the heterotrich "Stentor has a remarkable type of large nucleus resembling a string of beads . . . " (Buchsbaum, R. et al., 1971, Animals Without Backbones, 3rd ed., University of Chicago Press, Chicago, Ill.). These peculiarities of nuclear metamorphoses might even be linked in evolutionary time with the biochemistry of cells surviving in the pre-oxic environment insofar as they appear to grow in embryos, adenomas and adenomas in local milieu that would be expected to be oxygen poor prior to neo-vascularization. Warburg's discovery that amino acids provide the oxygen reducing equivalents for ATP generation in embryos and tumors suggests selection of a phenotype that treats oxygen as the limiting nutrient.

The heteromorphic nuclear morphotypes (e.g., bell-shaped nuclei) thus appear to represent a three-fold physiological nexus uniting evolutionary biology, embryogenesis and oncogenesis. To the genetic cycle of meiosis and mitosis, symmetrical and asymmetrical amitotic stages of lineal descent between the mitotic divisions of the post-fertilization period and the mitotic divisions that create most of the cellular mass of an animal must now be added.

The present invention is specifically directed to methods of classifying cell types based on nuclear morphology, their involvement in symmetrical and asymmetrical amitoses and their association in multicellular aggregates. For example, a tissue sample obtained from a mammal can be classified based on the presence or absence of heteromorphic nuclear morphotypes (e.g., bell-shaped nuclei, cigar-shaped nuclei and bullet-shaped nuclei). As FIG. 1 demonstrates, heteromorphic nuclear morphotypes are present in different stages of development and also at different stages of tumor development. For example, bell-shaped nuclei are found in fetal samples, rarely in adult samples, and prevalently in adenoma and adenocarcinoma samples. This supports the notion that heteromorphic nuclear morphotypes can be used to identify fetal juvenile stem cells as well as neoplastic stem cells in adult tissues. These results demonstrate a previously undocumented link between fetal stem cells and cancer stem cells. Thus, heteromorphic nuclear morphotypes that are indicative of fetal juvenile stem cells in fetuses are also indicative of cancer stem cells in adult tissues.

In addition, FIGS. 2A-2C (see Original Patent) show that heteromorphic nuclear morphotypes align in tube-like structures. As these tube-like structures are present in fetal samples and contain heteromorphic nuclear morphotypes (e.g., bell-shaped nuclei, cigar-shaped nuclei and bullet-shaped nuclei), the tube-like structures themselves can be used as indicia of fetal juvenile stem cells or neoplastic (e.g., tumor or cancer) stem cells in adult tissues.

Alternatively, the arraying of heteromorphic nuclear morphotypes into syncytia as shown in FIG. 5A-5E (see Original Patent) is indicative of differences between adenomas and adenocarcinomas. Therefore, the arrangement of nuclear morphotypes in multinuclear structures in a tissue sample can be used to differentiate among neoplastic samples at different stages of disease.

As heteromorphic nuclear morphotypes have been observed in other adult tissues and tumor samples (e.g., liver), one of skill in the art would recognize a preneoplastic or neoplastic lesions based on the presence of heteromorphic nuclear morphotypes in any adult tissue.

The present invention is also directed to methods for identifying anti-tumorigenic agents based on the appearance or disappearance of heteromorphic nuclear morphotypes specifically associated with preneoplastic or neoplastic tissues (e.g., bell-shaped nuclei, cigar-shaped nuclei and bullet-shaped nuclei). Candidate anti-tumorigenic agents can be screened, for example, in vivo in particular animal models (e.g., mammalian models, e.g., rodents such as, for example, mice or rats). Candidate anti-tumorigenic agents can be screened, for example, in clinical studies to discover if a trial regimen actually destroys the tumor stem cell component as opposed to the non-stem cell population that constitutes >99% of the cells in a tumor. With the process described herein candidate anti-tumor agents or tumor prevention agents can be screened in experimental animals using transplants from human tumors or tumors that arise de novo in experimental animals. Thus, agents that kill or interfere with the symmetrical or asymmetrical divisions of cells with bell-shaped nuclei in cell culture would be recognized as candidates for tumor prevention or therapy in patients.

Alternatively, anti-tumorigenic agents can be screened in vitro or ex vivo (e.g., in cultured samples where nuclear morphologies are maintained). Methods for preserving tissues in primary cultures for extended periods of time are known in the art. Anti-tumorigenic agents can be identified in cultured samples where heteromorphic nuclear morphotypes are preserved. Thus, if such a cultured sample is treated with a candidate anti-tumorigenic agent and the prevalence of heteromorphic nuclear morphotypes is diminished, then the candidate anti-tumorigenic agent would be expected to be useful in treating tumors in patients in vivo.
 

Claim 1 of 19 Claims

1. A method for identifying a stem cell of interest or multinuclear syncytium of interest, wherein the syncytium of interest comprises one or more stem cell nuclei and, wherein the cell or syncytium is obtained from or contained in a cell culture, pre-neoplastic lesion, tumor sample or tissue sample, wherein the stem cell of interest or syncytium of interest is identified by visualizing nuclear morphology, wherein the stem cell or syncytium of interest comprises a heteromorphic nuclear morphotype.
 

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