Colony assays

Clonogenic assays, where a single cell gives rise to a recognizable colony, are selective, since they can measure minority populations; the assays are specific, quantitative, qualitative and provide information about the proliferative and differentiation capacities of the cells of origin. Each of these advantages of colony assays has limitations. The most severe limitation of the method is that analysis of cellular composition to determine progenitor properties shows only what the cell of origin did during clonal expansion, but not what it might do if conditions were improved or the time of observation prolonged.

Colonies in the spleen (CFU-S)

The first colony assay depended on the capacity of about one in 10³ normal mouse marrow cells to give rise to a discrete nodule in the spleen of a heavily irradiated recipient animal.¹ The number of such nodules, consisting of differentiating blood cells, was linearly related to the number of marrow cells injected after heavy irradiation; counting spleen colonies was, therefore, a quantitative measurement of a single entity in marrow, originally called a colony-forming unit or CFU. The method became generally useful with the demonstration that each colony was a clone, derived from a single cell.² Proof that a CFU was a stem cell came from experiments where retransplanted primary colonies were found to contain new CFU.³ As well as extensive proliferative capacity, CFU could differentiate and also undergo self-renewal, the defining properties of stem cells.

The spleen colony assay contributed to radiobiology, hematology and transplantation. The experiments that found new CFU in spleen colonies showed that the distribution of new CFU among spleen colonies was highly skewed.^4,5 Stochastic processes were known to be mechanisms for generating such distributions. Such a mechanism was postulated for growth and differentiation during spleen colony formation. CFU were considered to have two contrasting fates; a stem cell might renew itself giving rise to two daughters that retained stem cell properties; this outcome was called 'birth'. Alternatively, a stem cell might enter into the process of differentiation; as a consequence it would lose the property of self-renewal and be lost from the stem cell population. This outcome was called 'death'. The model proposed that 'birth' and 'death' occurred at random during clonal expansion, with a fixed probability of each fate.⁶ The 'death' fate can no longer be equated only with differentiation. Much attention is now given to apoptosis, an active genetically regulated process that is recognized by orderly cutting of DNA to form a ladder that can be seen on appropriate gels.⁷

Many were not comfortable a lax mechanism for hematopoietic regulation. A more precise regulation came from experiments using genetically anemic mice of genotypes W/W^v and Sl/Sl^d. These are sterile, have a macrocytic anemia and extreme sensitivity to the lethal effects of ionizing radiation.⁸ This latter property provided the reason for using the spleen colony assay to study the genetically anemic mice, since CFU had been shown to be responsible for hematological recovery after ionizing radiation. Marrow from mice of genotype W/W^v was transplanted into heavily irradiated normal compatible recipients. Macroscopic colonies were not seen, indicating that the lesion at the W locus was preventing the proliferation of CFU.⁹ When the same experiment was repeated using Sl/Sl^d donors, there was extensive colonyformation. In contrast, when irradiated Sl/Sl^d mice were used as marrow recipients, no hematopoiesis was found in either spleen or marrow. The conclusion was that the gene at the Sl locus controlled an organ environmental factor that was required for marrow engraftment. Marrow from Sl/Sl^d mice could be transplanted into unirradiated W/W^v animals, where the stem cells functioned well and cured the anemia of the W/W^v recipients.¹⁰ These studies of regulation showed that intact genes at the W and Sl loci are needed for colony formation; but, when these requirements are met, clonal expansion is stochastic. Stability comes from fixed 'birth': and 'death' probabilities and by the averaging effects of normal polyclonal hematopoiesis.

The restriction of cells in colonies to a single splenic space gave rise to the question whether, at longer times, the progeny of a single CFU could repopulate the whole hematopoietic system of a recipient. Many methods of cell separation and phenotypic characterization combined with functional assays have been used to address the question. Unequivocal results were obtained in experiments where cells were infected with retroviruses that had unique integration sites; these then served as clonal markers that could be used to follow the histories of clones following marrow transplantation. After a year, cells of the same clones were identified in both myelopoietic and lymphoid organs. It follows that stem cells must exist that can differentiate into both of the main divisions of hematopoiesis. The experiment did not show whether such cells were also able to form spleen colonies.¹¹

The issue of the earliest stem cells was addressed by Irving Weissman and his collaborators who used phenotypic characterization to achieve a thousand-fold purification of stem cells; these have the phenotype c-kit^highThy^lowLin-Sca⁺.^12,13,14 Functional assays of such populations provided evidence of capacity to differentiate into both myeloid and lymphoid lineages, as might be expected from a reconstituting cell. Others have used cell separation procedures to prepare fractions enriched for progenitors recognized by colony assays and other fractions with capacity for long-term reconstitution.¹⁵ In these reports, populations with long-term reconstituting ability have always contained a very few cells able to form spleen colonies. Perhaps, reconstituting cells are stem cells that have extensive proliferative capacity because of a very short proliferative history.

Colonies in culture

A major advance was made when Bradley and Metcalf¹⁶ in Australia, and Pluznik and Sachs in Israel¹⁷ independently discovered ways to grow hematopoietic progenitors in culture under conditions that allowed colony formation. Two conditions were required: first, the culture medium had to be made viscid by the addition of agar or methylcellulose; second, a source of stimulator was required, provided either as a feeder layer or as supernatants from cell cultures. Using these two culture features, several systems were developed for clonogenic assays for progenitors in the three lineages of myelopoiesis and the two lineages of lymphopoiesis. The nomenclature was revised as new assays were described. CFU was retained, but modified to indicate each colony type. (reviewed by Metcalf¹⁸). Progenitors of late erythropoietic cells were called CFU-E.^19,20 Earlier cells of the lineage were recognized as the source of groups of smaller CFU-E colonies; these were called 'burst-forming units' or BFU-E.²¹ Methods for the three myelopoietic lineages were complete with the development of a clonogenic assay for megakaryocytes.^22,23 Clonogenic methods for B and T lymphocytes were also described.^24,25

Colony formation in culture depends of the presence of bioactive stimulators, colony-stimulating factors or CSF. The rapid development of methods for studying molecules were quickly applied to CSFs. One such, erythropoietin, was already well known, as a protein made in kidney and essential for the final stages in the maturation of red blood cells.^26,27 The gene encoding erythropoietin was soon cloned; its availability made it possible to make large quantities of erythropoietin, not only for research but also for clinical use.^28,29 Growth factors for cells in the myelopoietic and megakaryocytic lineage were unknown before culture assays were developed; soon genes encoding them were identified; genes for CSFs active on early progenitors, with bilineage progeny, IL-3³⁰ and GM-CSF³¹ were found to have common features and to act synergistically.³² The gene for CSF for later granulopoietic progenitors (G-CSF) was also cloned.^33,34 The assay for megakaryocytic colonies came later; its regulator, thrombopoietin, was also cloned. These growth factors proved to be ligands for cell surface receptors. Clones for genes encoding these receptors were also soon obtained. The receptor for thrombopoietin proved to be a receptor proto-oncogene, Mpl.^35,36,37

Mpl was not the first proto-oncogene to be recognized as a cell membrane receptor. C-kit is encoded by a gene at the W locus in the mouse (see earlier); when it was found to be the genetic source of a membrane receptor, the obvious conclusion was reached that c-kit was an important growth regulator.^{38,39,40,41,42} Many groups searched for the kit ligand, reasoning from the previous genetic studies that it should be the product of Sl locus. Success was soon achieved; the ligand was found, the clone isolated, its relation to Sl established and its functions as a growth regulator.^{43,44,45,46,47}

The geneticall-regulated control mechanism, expected from the finding with mice of genotypes W/W^v and Sl/Sl^d, was amply documented by studies of growth factors and their receptors. Ligands bind to receptors, which then institute signaling, where the information is carried by a series of proteins, that may be phosphorylated on tyrosine, serine or methionine by kinases or the phosphorus removed by dephosphorylase. The pathways are overlapping at several levels, including receptors, which may be common to more than one ligand,⁴⁸ and the protein networks that carry information.^49,50,51 Similar and overlapping mechanisms also regulate apoptosis. This 'death' probability may be initiated by damage to DNA or through the RAF receptor on the cell surface. The effectors of apoptosis, highly analogous to the CED genes in C. elegans,⁵² are proenzymes that are activated by caspases. Mitochondria are major central points, since the apoptosis pathways alter their membrane potentials and allow the liberation of effectors such a cytochrome C.⁵³ Apoptosis is regulated by a series of proteins that protect from death^54,55 and others that favor it.^56,57 All these data show that the cytoplasm is filled with proteins that are part of an interlocking three-dimensional information network. Yet, the cell appears to respond to the network with a limited number of binary outcomes including division or rest, self-renewal or differentiation, apoptosis or recovery. In a general sense, these outcomes may be characterized by 'birth' or 'death'. The stochastic model of clonal expansion, described earlier, balances the randomness of binary outcomes with the concept of fixed probabilities. It may be that the information network has, as one of its function, the establishment and maintenance of these probabilities.

Myeloblastic leukemias

The leukemic cells in chronic myeloblastic leukemia (CML) were proven to be clonal when the Philadelphia chromosome was found in all the three myelopoietic lineages.⁵⁸ Using sex-linked isoenzymes of glucose-6-phosphate dehydrogenase (G6PD) as clonal markers, Fialkow et al⁵⁹ confirmed the clonal nature of CML and showed that acute myeloblastic leukemia (AML) was a clonal disease, beginning as a transformation in a very early progenitor cell. In some instances the cell of origin was a pluripotent stem cell, while in others the data were compatible with transformation of an early cell committed to a single.^60,61 Other diseases, including Polycythemia rubra vera and ideopathic myelofibrosis also were found to be clonal.^62,63 The common properties of these clonal hemopathies (reviewed by McCulloch and Till⁶⁴) include clonal dominance and repression of coexisting normal populations. Cells from patients with the myeloblastic leukemias will form colonies in culture, using the methods developed for normal cells. With time, methods were developed that were more selective for abnormal blast cells (see below); these were used rather than techniques that allowed the development of cells of widely variable levels of differentiation.

Abnormal karyotypes in myeloblastic leukemias

Chromosomal abnormalities are common in the myeloblastic leukemias; they may be considered to fall into two categories. First, there are chromosome abnormalities that define specific diseases. The Philadelphia chromosome is found in more than 90% of cases of CML. It is now known to be a reciprocal translocation between chromosome 9 and 22⁶⁵ and is diagnostic of CML. The breakpoint on chromosome 22 is limited to a small region, termed the breakpoint cluster or bcr.⁶⁶ An oncogene, ABL, originally isolated from Abelson murine leukemia virus, is part of the translocation from 9 to 22, which encodes a fusion protein, bcr/abl. This protein is a tyrosine kinase, with enhanced activity. Transfer of the bcr/abl gene either in vivo or in vitro causes abnormal stem cell proliferation.⁶⁷ These findings make it highly likely that bcr/abl is the immediate cause of CML. A 15/17 translocation is characteristic of a subgroup of AML, acute promyelocytic leukemia (APL).⁶⁸ The translocation brings the retinoic acid receptor (RAR) close to the gene for a protein myl. The result is a novel fusion protein,⁶⁹ which confers on the cells an increased sensitivity to all trans retinoic acid (ATRA); APL cells exposed to ATRA in culture lose clonogenicity and appear to differentiate to mature granulocytes.⁷⁰

The second category of chromosomal abnormalities are those that are related to prognosis. Patients whose AML cells have inv16 or t8;21 have a significantly greater chance of entering remission, while those trisomy 8 and deletions of chromosomes 5 and 7 are less likely to respond to treatment.⁷¹ These karyotypic findings are the most effective predictors of response in cohorts of AML patients treated with standard chemotherapeutic regimens, excluding APL.

The blast cells of AML

A minority of AML blasts from peripheral blood or marrow will form colonies in culture under conditions very similar to those used for normal progenitors.^72,73,74 The colonies contain cells that have the same morphology of the blasts from which they were derived. Blast colonies were shown to contain new blast progenitors, although their numbers were small and varied greatly from in colonies from the cells of different patients. Nonetheless, their presence was evidence of the fact that clonogenic blast cells had the property of self-renewal and therefore could be considered as stem cells.

Association of blast characteristics with choice of treatment or outcome

The blast stem cells that were clonogenic in culture were seen to be appropriate targets for treatment. The research priority was to use the method to find blast properties that were either directive of treatment or of prognostic value. A striking feature of the assay was that colony formation almost always required the presence in the cultures of the same factors that were active on normal hematopoietic progenitors, including the kit ligand (stem cell factor), IL-3, GM-CSF and G-CSF. There was great clone variation in the response to factors, and synergism was often seen when more than one was added to the cultures.^75,76,77,78 AML appeared to be a dependent neoplasm. The possibility considered was that response to growth factors might have a prognostic value. When blast growth was evaluated by measuring uptake of ³HTdR about half of blast cell populations did not need factors to incorporate ³HTdR into DNA; an association was found between clinical outcome and this stem cell autonomy.⁷⁹ In contrast, when colony formation in culture was used, very few autonomous populations were seen, and no association emerged between response and survival.⁸⁰

Self-renewal of clonogenic blast stem cells might be expected to predict for the clinical outcome. Self-renewal was measured by replating blast cells selected either from individual colonies or from pools. The results was expressed as secondary plating efficiency or PE2. Since there was wide patient-to-patient variation in the value of PE2, it was feasible to look for an association with response to treatment. Such an association was found in several laboratories.^81,82,83 The result provided evidence for the biological importance of clonogenic blast cell self-renewal; the correlation was never strong enough to be useful in predicting the outcomes of individual patients.

The blast clonogenic assay was quantitative and could be used to measure dose–response curves for blast populations treated with chemotherapeutic drugs. Often the resulting dose–response curve could be fitted with a simple negative exponential, although more complex relations, sometimes with a plateau, were sometimes encountered.^84,85 Regardless, sensitivity may be described as the dose of drug required to reduce survival of colony formation to 10% of control (D10). Associations between D10 and response were often found, although, as in the case of the self-renewal measurements, the correlations were never strong enough to be used to select chemotherapy.

The weak and clinically unimportant association between culture characteristics of blasts and choice treatment or outcome was in striking contrast with the value of the 15/17 translocation in APL and the 9/22 translocation in CML. The fusion protein joining RAR and myl, associated with increased cytotoxic response to ATRA in culture, provided an explanation for the remarkable clinical response to ATRA, with the disease often going into complete remission.^86,87 This early evidence was confirmed in large trials. While often APL patients treated with ATRA alone relapsed, the addition of an anthracycline drug to the regimen often resulted in long-term survival.⁸⁸

The breakpoint cluster region of the 9/22 CML translocation, encodes a fusion protein, bcr/abl, a tyrosine kinase, with enhanced activity. Transfer of the bcr/abl gene either in vivo or in vitro causes abnormal stem cell proliferation. The molecular pathology of CML was the basis for a large program with the aim of finding a drug that bound to an important component of the fusion protein, bcr-abl. The tactic was to seek tyrosine kinase inhibitors; a compound 2-phenylaminopyrimidine, with some capacity to inhibit protein kinase C was chosen for further work, analyzing structure/function relations between chemically related compounds. Eventually one, STI571 was found that was an excellent tyrosine kinase inhibitor; culture experiments showed that STI571 was very active in decreasing colony formation by cells with the 9:22 translocation, while normal cells were not affected. Its mechanism appeared to be binding to the ATP binding site on the bcr-abl protein. This mechanism was confirmed by three-dimensional structural studies that showed the position of ATP binding cleft and the localization of STI571 to it.⁸⁹

The preclinical development of STI571 quickly led to phase I/II clinical trials, based on patients who had failed while on the standard treatment with interferon. In this early work, many of these end-stage patients entered remission and, in some, the Philadelphia chromosome-positive cell population was eliminated. Quickly, large trials were organized; these demonstrated the value of the drug in chronic phase, accelerated phase and blast phase of CML. In contrast with cytotoxic chemotherapy, very few side effects were encountered and these were not serious enough to require that the treatment be stopped. With time, relapses were seen; this experience, like that with ATRA in the treatment of APL, makes it reasonable that STI567 will be more effective if combined with other antileukemic drugs such as cytosine arabinoside or an anthracyline. Tissue culture experiments with these drugs have already shown both additive and synergistic effects.⁹⁰

Stem cell plasticity

Until recently, it was considered that stem cells functioned only in systems that depended on proliferation and differentiation; these obligatory renewal systems include hematopoiesis, epithelial mucosa and skin. Other systems respond to injury by divisions in cells already differentiated. For example, following partial hepatectomy, mature liver cells divide and replace the parenchyma that was removed; in a similar fashion, renal tubule cells can resume proliferation in case of injury. These long-held ideas have now been challenged seriously by experiments that show that hematopoietic stem cells can give rise to liver cells,⁹¹ muscle and brain.^{92,93,94,95,96} These studies have recently been reviewed by Wulf et al.⁹⁷ Of particular note is that these apparently totipotent stem cells, oval cells of liver and satellite cells of muscle have surface markers, such as Sca and c-kit that are usually associated with hematopoietic stem cells.

The plasticity of stem cells remains controversial. The findings, from several laboratories are such as to make it reasonable to assume that adult stem cells exist with the potential to differentiate and produce the functional cells of several organs. Such cells might well be targets for carcinogenic events. It follows that malignancies may exist that arise as transformations in plastic stem cells. The phenotypes of such tumors might continue to reflect the very extensive differentiation potential of plastic stem cells. A candidate is the well-documented clinical presentation, metastases with unknown primary site.⁹⁸ The disease is not rare, occurring in 4–8% of cancers. The remarkable diversity of the tumors is illustrated in Figure 1. At first pathological examination, four histological diagnoses were made; after further tests, including looking for expression of disease-related antigens, many more cancer types were found, ranging from carcinomas, sarcomas and melanomas. Although not shown on the slide, teratoma was occasionally found. In many incidences, no specific subtype was identified. In a minority of patients, a primary tumor was eventually found either during life or at autopsy. One might ask what criteria were used to establish such a primary? Usually, a tumor with a histological appearance consistent with its site is called primary. If, as postulated, the disease began as a transformation in a totipotent stem cell, a tumor at a specific site with an appropriate morphology might not be primary; rather it may have grown where found by the more random process that established the sites of growths considered to be metastatic.

Figure 1.

The distribution of pathologically classified tumor types within metastases of unknown primary site. The first row shows diagnoses based on the first pathological examination. The second row shows the diagnoses made with antibodies, electron microscopy and molecular methods. Reprinted from,⁹⁸ with permission of the authors and publishers, the Massachusetts Medical Society, copyright 1993.

Full figure and legend (27K)

Conclusion

Clonogenic assays in vivo or in culture are powerful tools that have provided the basis for extending hematopoietic lineages to cells earlier than those recognized morphologically. The assays have important weaknesses; they show only what their cell of origin has done during clonal expansion rather than its full potential. In part, this limitation is inherent on the limited space occupied by a spleen colony or a colony in culture. A consequence is the continuing uncertainty about the earliest events in blood formation.

Colony assays have disclosed great diversity in both normal and leukemic hematopoiesis. Diversity is generated during clonal expansion from normal stem cells. Leukemic populations in patients are clones; diversity is evident in the very great patient-to-patient variation seen in clonal characteristics and the outcome of treatment. In both instances, diversity is a challenge for understanding regulation. Two very different control systems appear to operate together. The first is stochastic, with self-renewal or differentiation occurring at random at each cell division. In contrast with this lax control, a three-dimensional complex interlocking network of information-bearing proteins operate within the cell. The outcome of regulation is one of the two alternative outcomes. The suggestion is advanced that a stochastic mechanism sets the outcome, but that probabilities emerge from the protein network.

Diversity increased markedly with findings from several laboratories that showed that adult tissues contained stem cells that could repopulate both marrow and other apparently stable tissues.

Making the reasonable assumption that adult stem cells exist with great potential for both proliferation and differentiation, the question was asked whether any known clinical malignancy might start as a transformation in such stem cells. The hypothesis was offered that the entity called metastatic cancer with unknown primary might begin in stem cells exhibiting plasticity. The proposition is made in the hope that laboratory and clinical research might be undertaken to test the hypothesis. If the idea that adult stem cells are transformed and then grow in many different sites is supported, then a clinical entity will become a malignancy with diversity that mirrors the capacity of its cell of origin to populate several different tissues. If support is not found, the research will certainly yield new insights into both adult stem cells with plasticity and metastases with unknown primary site.

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