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Results for “clonal modeling”

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PubMed2001

Transplanted long-term cultured pre-BI cells expressing calpastatin are resistant to B cell receptor-induced apoptosis.

Long-term cultured pre-B cells are able to differentiate into immunoglobulin (Ig)M-positive B cells (IgM(+) cells) when transplanted into severe combined immunodeficient (SCID) mice. Based on previous studies, here we report the development of a reconstitution assay in nonobese diabetic/SCID (NOD/SCID) mice using pre-B cells, which allows us to study the role of calpains (calcium-activated endopeptidases) during B cell development as well as in B cell clonal deletion. Using this model, we show that calpastatin (the natural inhibitor of calpains) inhibits B cell receptor-induced apoptosis in IgM(+) cells derived from transplanted mice. We thus hypothesize an important function for calpain in sculpting the B cell repertoire.

Animals↗

PubMed2000

TCR v(beta) repertoire restriction and lack of CDR3 conservation implicate TCR-superantigen interactions in promoting the clonal evolution of murine thymic lymphomas.

Thymic lymphoma development is a multistage process in which genetic and epigenetic events cooperate in the emergence of a malignant clone. The notion that signaling via TCR-ligand interactions plays a role in promoting the expansion of developing neoplastic clones is a matter of debate. To investigate this issue, we determined the TCR V(beta) repertoire of thymic lymphomas induced in AKR/J mice by either endogenous retroviruses or the carcinogen, N-methyl-N-nitrosourea (MNU). Both spontaneous and MNU-induced lymphomas displayed restricted V(beta) repertoires. However, whereas V(beta)6, V(beta)8 and V(beta)9 were expressed by a greater than expected frequency of MNU-induced lymphomas, V(beta)8, V(beta)7, V(beta)13 and V(beta)14 were over-represented on spontaneous lymphomas. The dissimilar TCR V(beta) profiles indicate that different endogenous ligands promote neoplastic clonal expansion in untreated and MNU-treated mice. Although the nature of these ligands is not clear, the lack of conservation in TCR beta chain CDR3 regions among lymphomas that express the same V(beta) segment suggests that endogenous superantigens (SAG), as opposed to conventional peptide ligands, are likely to be involved in the selection process. The biased representation of lymphomas expressing V(beta)6-, V(beta)7- and V(beta)9-containing TCRs that recognize endogenous SAG is consistent with this hypothesis. The finding that Bcl-2 is expressed at high levels in spontaneous and MNU-induced lymphomas suggests that preneoplastic thymocytes may be resistant to SAG-induced clonal deletion. A working model is presented in which preneoplastic clones expressing TCRs that recognize endogenous SAG are selectively expanded as a consequence of sustained TCR-mediated signaling.

Animals↗

PubMed1993

Effective population size in organisms with complex life-histories.

A method for determining effective population size in organisms with arbitrarily complex life-histories, including clonal reproduction, is developed. A coalescent model that focuses on the time at which two alleles last shared a common ancestor leads to a method for determining an inbreeding effective population size. Comparisons with previously developed approximate methods for haploid age-structured models show close, but not exact, agreement. A diploid population with both clonal and sexual reproduction is considered; examples from published demographic studies are used to show the effects of within-individual versus between-individual coalescence times and different definitions of generation time in calculating effective population size. These examples show relatively small differences in effective population sizes calculated using within- and between-individual coalescence times; however, in one case, different definitions of generation time lead to a large change in ratios of effective population size to census size (Ne/N). The Ne/N calculated for clonally reproducing organisms in this paper seem to be substantially smaller than published values for organisms lacking clonal reproduction.

Age Factors↗

PubMed2001

The initiation of B cell clonal expansion occurs independently of pre-B cell receptor formation.

Current models of B cell development posit that clonal expansion occurs as a direct result of Ig H chain expression. To test this hypothesis, we isolated a population of early B cells in which H chain recombination is initiated and assessed V(H)DJ(H) rearrangements in both cycling and noncycling cells. We found that actively dividing cells within this population are enriched for H chain rearrangements that are productive when compared with their counterparts in G(0)/G(1), apparently supporting a role for H chain expression in initiating early B cell division; entrance into the cell cycle was accompanied by V(H) gene-dependent H chain selection. However, we also identified a phenotypically identical population of actively cycling early B cells in the absence of H chain expression in recombination activating gene knockout mice. In addition, actively cycling early B cells could be detected in pre-B cell receptor (pBCR)-negative lambda5 knockout mice, but we found no evidence for V(H)-dependent H chain selection in this population. Given these results, we suggest that the initiation of clonal expansion, at this early stage in B cell development, occurs independently of H chain expression. Although the cycling cell pool is enriched for pBCR-positive cells in mice expressing surrogate L chain, pBCR formation is not required for the initiation of cell division.

Animals↗

PubMed2001

Negative selection during the peripheral immune response to antigen.

Thymic selection depends on positive and negative selective mechanisms based on the avidity of T cell interaction with antigen-major histocompatibility complex complexes. However, peripheral mechanisms for the recruitment and clonal expansion of the responding T cell repertoire remain obscure. Here we provide evidence for an avidity-based model of peripheral T cell clonal expansion in response to antigenic challenge. We have used the encephalitogenic, H-2 A(u)-restricted, acetylated NH(2)-terminal nonameric peptide (Ac1-9) epitope from myelin basic protein as our model antigen. Peptide analogues were generated that varied in antigenic strength (as assessed by in vitro assay) based on differences in their binding affinity for A(u). In vivo, these analogues elicited distinct repertoires of T cells that displayed marked differences in antigen sensitivity. Immunization with the weakest (wild-type) antigen expanded the high affinity T cells required to induce encephalomyelitis. In contrast, immunization with strongly antigenic analogues led to the elimination of T cells bearing high affinity T cell receptors by apoptosis, thereby preventing disease development. Moreover, the T cell repertoire was consistently tuned to respond to the immunizing antigen with the same activation threshold. This tuning mechanism provides a peripheral control against the expansion of autoreactive T cells and has implications for immunotherapy and vaccine design.

Amino Acid Sequence↗

PubMed1995

Septal cell lines derived from the trisomy 16 mouse: generation, characterization, and response to NGF.

The trisomy 16 mouse is a genetic model of Down syndrome. Clonal cell lines were developed from trisomic as well as euploid embryonic mouse septal cells by introduction of thermolabile large T antigen mutant of SV 40. The cell lines underwent morphological differentiation at the non-permissive temperature and in response to a differentiating agent. Immunocytochemical staining indicated that cells of neuronal lineage were immortalized. The addition of beta-nerve growth factor (100 ng/ml) increased the survival rate of a trisomy cell line in differentiated state, as measured by Trypan blue exclusion. These cell lines may prove useful in studies of neuronal abnormalities in this mouse model of Down syndrome.

Animals↗

PubMed1989

Neurovirulence of two clonally related herpes simplex virus type 1 strains in a rabbit seizure model.

Herpes simplex virus type 1 (HSV-1) strains vary widely with regard to neurovirulence, but their tropism for specific central nervous system structures and their ability to induce seizures are poorly defined. We have used the clonally related +GC and -GC strains of HSV-1 to define the pathophysiological basis of neurovirulence in a rabbit model. Following intranasal inoculation, +GC infection was nearly uniformly fatal while -GC infection was asymptomatic. The +GC infected animals developed electroencephalographic (EEG) abnormalities which preceded severe motor seizures. Tropism of the +GC strain for specific CNS nerve centers and the expression of viral antigens within them correlated with its virulence. Although both viruses invaded and replicated within the brain, +GC replicated to slightly higher titers and expressed more abundant viral antigen than -GC. The relatively less efficient replication of -GC appeared to correlate with its temperature-sensitive phenotype in vitro. Both +GC and -GC antigens were found in cerebral cortical layers IV-VI, and in several central nervous system trigeminal and olfactory system structures. However, +GC spread more completely throughout the brain to involve the amygdala, nucleus accumbens, several brainstem nuclei and the locus ceruleus. The +GC antigens were also found in cerebral cortical layer I of animals that developed seizures. These results indicate that the ability of HSV-1 to induce electrophysiologic brain abnormalities is associated with its ability to replicate within specific brain nerve centers.

Animals↗

PubMed1992

A review of the role of anti-opioid peptides in morphine tolerance and dependence.

Studies on the mechanisms of tolerance and dependence have mostly focused on changes at the receptor level. These experiments, conducted with model systems ranging from clonal cell lines to whole animals, have identified a number of important adaptive mechanisms which occur at the receptor level. However, none of these adaptive mechanisms can completely account for the phenomena which serve to define the state of morphine tolerance and dependence, especially the observation that as an animal becomes more tolerant to morphine, less naloxone is required to trigger withdrawal. The data reviewed in this paper provide strong support for the hypothesis that the brain synthesizes and secretes neuropeptides which act as part of a homeostatic system to attenuate the effects of morphine and endogenous opioid peptides. According to this model, administration of morphine releases anti-opioid peptides (AOP), which then attenuate the effects of morphine. As more morphine is given, more AOP are released, thereby producing tolerance to the effects of morphine. Cessation of morphine administration, or administration of naloxone, produces a relative excess of anti-opioid, which is in part responsible for the withdrawal syndrome. Since endogenous and exogenous antagonists might together produce synergistic effects, less naloxone might be required to trigger withdrawal in the presence of higher levels of AOPs. Although the study of AOP is in its infancy, a deeper understanding of the central nervous system (CNS) anti-opioid systems may lead to new treatments for chronic pain, substance abuse, and psychiatric disorders.

Animals↗

PubMed2004

Cutting edge: heterozygote advantage in autoimmune disease: hierarchy of protection/susceptibility conferred by HLA and killer Ig-like receptor combinations in psoriatic arthritis.

Functionally relevant combinations of HLA and killer Ig-like receptor (KIR) genotypes influence resistance to several diseases in humans. Analysis of genetic data from such studies is challenging because it involves multiple linked and unlinked loci that exert their influence in an epistatic manner. We previously reported that subjects with certain activating receptors were susceptible to developing psoriatic arthritis (PsA), an effect that was strongest when HLA ligands for corresponding homologous inhibitory receptors were missing. In this study, we present a novel model in which susceptibility to PsA is determined by the overall balance of activating and inhibitory composite KIR-HLA genotypes. This model fits our knowledge of clonal NK cell expression of KIR and regulation of NK cell activity better than does the previous model, as reflected in a robust trend for increasing susceptibility to PsA with more activating genotypes. These data emphasize the remarkable influence of KIR/HLA combinations on this disease.

Arthritis, Psoriatic↗

PubMed2000

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on initiation and promotion of GST-P-positive foci in rat liver: A quantitative analysis of experimental data using a stochastic model.

We use a stochastic model describing initiation and clonal growth of altered cells to analyze data from an initiation-promotion hepatocarcinogenesis experiment in female Wistar rats. Starting at 7 weeks of age, the animals were treated for 10 days with the initiating agent diethylnitrosamine (DEN, 10 mg/kg body wt per day). After a 10-week resting period, the animals were treated either with corn oil or with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via biweekly sc injections of 1.4 microg/kg body wt of TCDD dissolved in corn oil. Groups of four or five animals were euthanized 3, 17, 31, 73, and 115 days after start of TCDD/corn oil treatment. The data analyzed consist of the number and sizes of GST-P-positive focal transections at various time points. By fitting the model to the data, we estimate the rates of initiation, cell division, and cell death during different time periods of the experiment. The model estimates of cell kinetic parameters are consistent with directly made experimental observations of cell division and cell death. The model predicts that DEN-induced initiation of GST-P-positive cells is highly protracted in controls and TCDD-treated animals alike. We also find that TCDD interferes with the normal rate at which cells with (DEN-inflicted) DNA damage are converted into cells expressing the GST-P-positive phenotype, suggesting a TCDD-mediated "acceleration" of the appearance of de novo GST-P-positive initiated cells from damaged precursor cells. Furthermore, the model predicts a significant reduction in the rate of apoptosis within the first 4 to 5 weeks of TCDD treatment, and after 10 weeks of TCDD treatment, but not in between.

Animals↗

PubMed1990

T47DCO cells, genetically unstable and containing estrogen receptor mutations, are a model for the progression of breast cancers to hormone resistance.

We postulate that one mechanism for the progression of breast cancers to hormone resistance involves the acquisition of mutant estrogen receptors (ER)4 by genetically unstable cell subpopulations. The T47D human breast cancer cell line may be a model for such progression, having sublines that are ER positive and estrogen responsive, ER positive and estrogen resistant, or ER negative. Also, T47D cells can be either hyperdiploid (HD) or hypertetraploid (HT) or persistently alternate between these states. T47DCO cells are a HD and ER-positive, but estrogen-resistant, subline of T47D cells that undergoes spontaneous tetraploidization. Such a stable variant, designated T47Dv, is 85% HT (Cancer Res., 49: 3943, 1989). We now show that single-cell clones derived from the mixed HD/HT T47Dv can be either HD or HT, and can be either estrogen responsive or estrogen resistant, for growth and for progesterone receptor regulation. To begin the study of ER in this model system of T47DCO and their derivatives, we have generated complementary DNA libraries from the parental HD T47DCO cells and have isolated three ER complementary DNA mutants. These include two frame-shift/termination mutants that would encode ERs truncated in the DNA-binding domain and in the hormone-binding domain and a third mutant with a large in-frame deletion spanning the hinge region and a part of the hormone-binding domain. If expressed, these mutant ERs would lack hormone-binding capacity and would be undetected by the anti-ER antibodies currently in clinical use. Genetic instability, when associated with mutant ERs in subpopulations of breast tumor cells, may provide the selective pressure leading to hormone resistance. T47DCO cells and their clonal derivatives provide a model for the systematic study of ER mutations and other mechanisms of hormone resistance in Stage IV breast cancer.

Amino Acid Sequence↗

PubMed1988

Molecular analysis of membrane gamma 2b heavy chain expression.

In order to study T cell regulation of B cell isotype differentiation we have developed a model system consisting of clonal populations of T and B cells. Using this system we have shown that the murine B cell lymphoma, 70Z/3, can be induced to express membrane IgG2b by exposure to a T cell hybridoma derived from the Peyer's patch (termed HAJ-3). The membrane bound IgG2b (mIgG2b) expression is associated with induction of gamma 2b-mRNA, but switch region rearrangement and C mu deletion does not occur. While LPS-stimulated 70Z/3 B cells also express considerable amounts of gamma 2b-mRNA they do not express detectable mIgG2b, indicating that T cell influence is necessary for the production of translatable gamma 2b-specific mRNA. Both the LPS and T cell induced gamma 2b mRNA transcripts lack VH sequences, implying that the surface IgG2b detected lacks a variable region. These findings lend support to a two step model of B cell isotype switching and provide evidence that T cells can regulate early events involved in B cell isotype differentiation.

Animals↗

PubMed2006

An etiologic model proposing that sporadic adult-onset carcinoma is extramedullary hematopoiesis.

This model proposes that primary carcinomatous tumors and almost all metastases are extramedullary hematopoietic tissue formed to compensate for reduced hematopoietic activity in the bone marrow. These marrow lesions are currently considered to be metastatic in origin, but as fibrosis and sclerosis are identifying features they are here equated to myelofibrosis. Myelofibrotic marrow is characterized by an increase in the number and size of vascular sinusoids. The increased blood flow suggested by this morphology, and observed in myelofibrosis patients, causes a rise in marrow pressure which may trigger the fibrosis. Specific carcinoma morphologies are equated to stages in endochondral bone and marrow formation and, as such, cancer cell identity varies with morphology. For example, infiltrating carcinomas of the breast consist of collagen and mucoid secreting cells in single file formation. This morphology is equated to the cartilagenous stage of marrow formation, when mesenchymal stem cells proliferate and differentiate into chondroblasts. In this model "infiltrating" cells arise in situ from stem cells located in the connective tissue. Tubular breast carcinoma, with its single layer of osteoblast-like carcinoma cells encircling small lumens and long branching tubules, is equated to the trabecular stage of marrow formation during which osteoblasts surround small pieces of calcified cartilage and begin secreting osteoid that will form the trabeculae. Lobular carcinoma in situ consists of cancer cell clusters separated by narrow clear spaces that, under high magnification, appear vascular. This morphology is equated to hematopoietic tissue in which primitive hematopoietic cells lie between anastomosing sinusoids. Similar cartilagenous, trabecular and hematopoietic morphologies can be found in carcinomatous tumors of most organs, but the nomenclature is variable. The hematopoietic carcinomas share numerous features with hematopoietic tissue including a structure composed of intermingled normoxic and hypoxic regions and a metabolism characterized by elevated levels of glycolysis. They also contain similar proportions of clonal cells. If this model is correct it necessitates a change in the treatment of carcinoma. If cancer cells are not the enemy, but desperately needed immature blood cells, and the medical problem is not the presence of tumors, but the inefficiency of this extramedullary hematopoietic tissue, then treatment should focus on increasing marrow hematopoiesis. As evidence suggests that the marrow lesion is the result of increased hydrostatic pressure this could be done by reducing blood volume. One way to accomplish this may be through the ingestion of ephedrine, as it is hypothesized to increase vascular tone.

Adult↗

PubMed1991

Experimental oncogene induced prostate cancer.

The mouse prostate reconstitution model exploits the ability of the fetal urogenital sinus to differentiate into a mature prostate when grafted under the renal capsule of an adult isogenic male host. By use of a recombinant retroviral vector, the ras and myc oncogenes are introduced singly or in combination into the fetal urogenital sinus--resulting in distinct phenotypes of prostatic pathology: dysplasia (caused by ras), hyperplasia (caused by myc) and frank carcinomas (caused by a combination of ras+myc). This unique experimental model creates in vivo conditions that mimic the natural initiation and progression of cancer. An expanded MPR protocol allows restricted retrovirus infection of the mesenchyme or epithelial compartments to evaluate paracrine activities. It enables almost unparalleled flexibility in addressing fundamental questions in prostate cancer. We have identified genetic variance in the susceptibility to tumour induction between two different strains of mice (mimicking the observation of racial variability in the predisposition to clinical prostate cancer). The MPR model supports data from other tumour models and implicates TGF-beta 1 and TGF-beta 3 as being strongly associated with tumour progression. Finally, with this model, we have established clonal prostate adenocarcinomas to study directly the affects of castration on gene expression. Not only are TGF-beta 1 and TGF-beta 3 mRNA levels increased in association with malignancy but they are also further enhanced by castration treatment. Based on these experimental studies, we believe that TGF-beta 1 and TGF-beta 3 expression strongly influences the progression of prostate cancer. This information will hopefully impact on the development of more effective therapy for this important malignancy.

Adenocarcinoma↗

PubMed1996

Evidence for a thymus-dependent form of tolerance that is not based on elimination or anergy of reactive T cells.

The avian embryo has provided an appropriate model to study the ontogeny of the primary lymphoid organs, thymus and bursa of Fabricius. By using the quail-chick marker system the embryonic origin of the highly intricate cell components which form these organs could be traced back to the initial endodermal, mesodermal and ectodermal germ layers. The timing and dynamics of the incoming and outcoming flows of hemopoietic cells which characterize their lymphopoietic activity could be revealed in both quail and chick embryos. This knowledge served as a basis for an investigation on the role of the epithelial component of the thymus (derived from the pharyngeal endoderm) on tolerance to tissue graft and, by extension, tolerance to self. When this work was undertaken, the prevailing view was that exposure of the developing immune system to foreign antigens in the embryo allows them to be assimilated to self components in the mature animal. In fact, this was found to be true for allogeneic grafts between MHC-distinct chickens, of certain tissues, such as for instance wing tissues. However, in heterospecific transplantations, i.e. when a limb bud was grafted from quail to chick embryos, the chick host acutely rejected the foreign limb soon after birth. In contrast, grafts of the quail thymic epithelial (TE) rudiment resulted in the development of a chimeric thymus in which the foreign epithelial component was not only tolerated but able to induce full tolerance of the grafted wing from the same donor. By monitoring the amount of quail TE implanted we showed in addition that only part of the peripheral T-cell population had to differentiate in the context of the quail epithelial cells to induce tolerance to quail tissues. This pointed to the generation in the thymus of regulatory T cells, coexisting with specific anti-quail reactive T cells, but able to inhibit them from reacting against the quail wing antigenic determinants. A mammalian model was then devised to further study this mechanism of tolerance that we have qualified as "dominant" by opposition to the current model based on either clonal elimination or anergy which can be considered as recessive or passive. Nude mice of MHC type A were grafted with TE of E10 type B embryos. They became reconstituted for T-cell function but tolerant for B skin allografts. Spleen cells from such tolerant animals injected to naive A nude mice reconstituted T cell function in the recipient and transferred the tolerance to B skin grafts. Reducing the number of donor cells resulted in the segregation of the two phenomena. For low numbers the recipients were restored but not tolerant, thus showing the coexistence in the tolerant donor of anti-B reactive T cells together with regulatory cells able to abolish their reactivity against B determinants. Other experiments demonstrated that TE-induced tolerance does not rely on clonal deletion or anergy. This was shown on systems where elimination of cells directed toward superantigens was screened. It turned out that tolerance to skin grafts and superantigen T-cell deletion are unrelated phenomena. These observations strongly suggest that tolerance to self results at least in part from the interplay between cells potentially harmful for self component and others which exert a strong control on their reactivity. The latter cell type depends upon interactions of thymocytes with the endodermal component of the thymus.

Animals↗

PubMed1996

Ligation of CD40 with soluble CD40 ligand reverses anti-immunoglobulin-mediated negative signalling in murine B lymphoma cell lines but not in immature B cells from neonatal mice.

Ligation of surface immunoglobulin (sIg) on certain murine B-lymphoma lines has been shown to initiate a programme leading to growth arrest and death of the cells by apoptosis. The cell lines WEHI 231 and CH33 which respond in this way to receptor cross-linking have phenotypic characteristics resembling those of immature normal B cells, and their responses have been taken to model those responsible for clonal deletion or anergy. Cross-linking of sIg on normal neonatal B cells has also been shown to inhibit their responsiveness to polyclonal activators. We have examined the ability of various co-stimuli to modify the response of growth-inhibitable B lymphoma lines to sIg cross-linking. Our findings indicate that cell-cell contact between cells of the WEHI 231 or CH33 lines and activated T cells rescues these cells from growth arrest and apoptosis. Cell-free supernatants from some T-cell lines were also protective although recombinant IL-4 had no effect. Analysis of the most effective signals and timing for inducing this protection suggested that it might, in part, be mediated by CD40 ligand (CD40L) expressed on or secreted by activated T cells. Using a soluble recombinant CD40L-CD8 fusion protein we have now shown that co-ligation of CD40 is sufficient to rescue WEHI231 and CH33 cells from anti-Ig-induced apoptosis. In contrast, the inhibitory effect of anti-Ig antibodies on the lipopolysaccharide (LPS)-driven proliferation of neonatal B cells was not relieved by co-ligation of CD40 with CD40L. These findings bring into question the usefulness of 'immature' B-cell lines as models for tolerance induction.

Animals↗

PubMed2006

Anatomy of T cell autoimmunity to myelin oligodendrocyte glycoprotein (MOG): prime role of MOG44F in selection and control of MOG-reactive T cells in H-2b mice.

Myelin oligodendrocyte glycoprotein (MOG) is an important myelin target antigen, and MOG-induced EAE is now a widely used model for multiple sclerosis. Clonal dissection revealed that MOG-induced EAE in H-2(b) mice is associated with activation of an unexpectedly large number of T cell clones reactive against the encephalitogenic epitope MOG35-55. These clones expressed extremely diverse TCR with no obvious CDR3alpha/CDR3beta motif(s). Despite extensive TCR diversity, the cells required MOG40-48 as their common core epitope and shared MOG44F as their major TCR contact. Fine epitope-specificity analysis with progressively truncated peptides suggested that the extensive TCR heterogeneity is mostly related to differential recognition of multiple overlapping epitopes nested within MOG37-52, each comprised of a MOG40-48 core flanked at the N- and/or the C-terminus by a variable number of residues important for interaction with different TCR. Abrogation of both the encephalitogenic potential of MOG and T cell reactivity against MOG by a single mutation (MOG44F/MOG44A), together with effective down-regulation of MOG-induced EAE by MOG37-44A-52, confirmed in vivo the primary role for MOG44F in the selection/activation of MOG-reactive T cells. We suggest that such a highly focused T cell autoreactivity could be a selective force that offsets the extensive TCR diversity to facilitate a more "centralized control" of pathogenic MOG-related T cell autoimmunity.

Amino Acid Substitution↗

PubMed2003

Determination of clonality of metastasis by cell-specific color-coded fluorescent-protein imaging.

It has been thought that metastases are clonal and originate from rare cells in primary tumors that are heterogeneous in genotype and phenotype. Recent studies using DNA array analysis challenge this hypothesis and suggest the genetic background of the host is the important determinant of metastatic potential implying that metastases are not necessarily clonal. Previous methods to determine clonality of metastasis used karyotype or molecular analysis that were complicated, thereby limiting the number of metastatic colonies analyzed and the conclusions that could be drawn. We describe here the use of green fluorescent protein-labeled or red fluorescent protein-labeled HT-1080 human fibrosarcoma cells to determine clonality by simple fluorescence visualization of metastatic colonies after mixed implantation of the red and green fluorescent cells. Resulting pure red or pure green colonies were scored as clonal, whereas mixed yellow colonies were scored as nonclonal. In a spontaneous metastasis model originating from footpad injection in severe combined immunodeficient mice, 95% of the resulting lung colonies were either pure green or pure red, indicating monoclonal origin, whereas 5% were of mixed color, indicating polyclonal origin. In an experimental lung metastasis model established by tail vein injection in severe combined immunodeficient mice, clonality of lung metastasis was dependent on cell number. With a minimum cell number injected, almost all (96%) colonies were pure red or green and therefore monoclonal. When a large number of cells were injected, almost all (87%) colonies were mixed color and therefore heteroclonal. We conclude that spontaneous metastasis may be clonal because they are rare events, thereby supporting the rare-cell clonal origin of metastasis hypothesis. The clonality of the experimental metastasis model depended on the number of input cells. The simple fluorescence method of determining clonality of metastases described here can allow large-scale clonal analysis in numerous types of metastatic models.

Animals↗