Patent Application
20090094703
The present invention generally relates to cells, cell lines and cell line culturing systems, and in particular, relates to novel medulloblastoma-forming cells.
Brain tumors are the second most common malignancy among children less than 20 years of age. Medulloblastoma is the most common malignant brain tumor arising in children, comprising 14.5% of newly diagnosed cases. Medulloblastoma originates in the cerebellum or posterior fossa and is the most common member of the family of cranial primitive neuroectodermal tumors (PNET). All PNET tumors of the brain are invasive and rapidly growing tumors that, unlike most brain tumors, spread through the cerebrospinal fluid (CSF) and frequently metastasize to different locations in the brain and spine.
Treatment of medulloblastoma includes surgical removal of tumour in combination with radiation and chemotherapy to increase the chances of disease-free survival. This combination may permit a 5 year survival in more than 80% of cases; however, aggressive treatment approaches, especially craniospinal irradiation, can harm the developing brain. It is hard to predict what dose of radiotherapy will be harmful in each individual child. On the other hand, the use of decreased dosages of radiation may not be sufficient to treat the tumour.
Over the past ten years, there has been increasing evidence that chemotherapy improves survival for children whose tumors cannot be fully resected, or have metastasized beyond the primary site at diagnosis. Studies attempting to delay and possibly even obviate radiotherapy in children, particularly those less than three years of age, are also underway using high-dose, multiple agent chemotherapy for administration immediately following surgery. Other studies are also underway involving chemotherapy directly within the cerebrospinal fluid.
Understanding the biology of medulloblastoma, its origin and what controls its growth is essential to the development of effective treatment protocols that obviate the disadvantages of current treatments, such as the adverse effects of radiation and side effects from non-specific treatments. It is known that human brain cancer growth is sustained by relatively rare therapy resistant cancer stem cells (CSC) both in vitro and in vivo. Although mouse tumours recapitulate many features of human cancer it is controversial whether these cancer models comprise a CSC hierarchy.
In order to advance the development of effective treatments for medulloblastoma, it would, thus, be desirable to develop a biological system which effectively mimics medulloblastoma in humans in order that novel therapies, such as chemotherapies, can be developed.
Novel medulloblastoma-forming cells have now been identified which are clonogenic. The identification of such clonogenic cells permits the establishment of cell lines and non-human animal models that effectively mimic medulloblastoma in humans and, thus, which are useful to provide in vitro and in vivo screens for therapeutics to treat human medulloblastoma.
Thus, in one aspect of the present invention, isolated medulloblastoma-forming clonogenic cells are provided.
In another aspect, an isolated medulloblastoma-forming cell line is provided comprising Nestin+Sox2+ medulloblastoma cells.
In another aspect, a non-human transgenic animal model is provided that has been transplanted with a cell line comprising medulloblastoma-forming clonogenic cells.
In a further aspect, a method of producing a cell line comprising medulloblastoma-forming clonogenic cells. The method comprises the steps of obtaining a sample of medulloblastoma cells and culturing the medulloblastoma cells in serum free conditions with EGF and FGF to establish a cell line.
In yet a further aspect, methods of screening candidate therapeutic compounds both in vitro and in vivo are provided using the foregoing cell lines and transgenic animal model, respectively.
These and other aspects of the invention will become apparent in the following detailed description and the drawings in which:
Isolated medulloblastoma-forming clonogenic cells are provided that may be used to prepare cell lines and transgenic non-human animal models that mimic human medulloblastoma.
The term “isolated” is used herein to refer to medulloblastoma-forming clonogenic cells which are substantially free from non-proliferative medulloblastoma cells with which they exist in vivo, for example, substantially free from other cells of the primary tumor from which they were derived.
The term “clonogenic” is used herein to refer to cells which possess the potential to proliferate and form a colony of cells.
The medulloblastoma-forming clonogenic cells of the present invention are characterized as Nestin+Sox2+ cells that exhibit long term proliferative potential, multilineage differentiation capacity and retain activated Hedgehog (Hh) and Notch signaling as evidenced, for example, by established assay, e.g. assay for β-galactosidase expression.
In one embodiment, the medulloblastoma-forming clonogenic cells may be Patched1 (Ptc1)-deficient, e.g. exhibits deficient ptc1 expression as a result of the ptc1+/− genotype. Patched1 is a receptor for Hedgehog signaling proteins. In another embodiment, the medulloblastoma-forming clonogenic cells may be deficient in tumour protein 53 (also known as protein 53 or p53), a tumour-suppressing transcription factor encoded by the TP53 gene. Tumour protein 53 (p53) deficiency refers to inclusion of either p53+/− and p53−/− alleles. In a further embodiment, the medulloblastoma-forming clonogenic cells are CD15+/−, e.g. expresses CD15 (3-fucosyl-N-acetyl-lactosamine), also known as Lewis x and specific embryonic antigen 1 (SSEA-1), is a stem cell marker.
The medulloblastoma-forming clonogenic cell line in accordance with the present invention may be obtained using a culturing protocol in which Patched 1 (Ptc1)-deficient medulloblastoma cells are incubated in serum free conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) for a period of time sufficient to result in enrichment of the clonogenic cells, for example, a period of time known in the art to be appropriate for cell culturing. The term “serum free” as used herein refers to the absence of any serous fluid including blood plasma. Using this protocol, a stable cell line is established which is viable through multiple passages, for example, at least 10 passages and preferably, greater than 25 passages, for example, greater than 40 passages.
An established medulloblastoma-forming cell line may be used in a method to screen candidate therapeutic compounds. Such a screening method includes the steps of culturing the cell line in the presence of a selected candidate compound for a sufficient period of time and then determining whether the candidate inhibits Hh or Notch signaling. This determination may be made by assaying the expression of Hh signaling targets, such as Gli1, Gli2 and/or Gli3, as well as the expression of Notch target genes such as Hes1 and Hes5. Decreased expression of an Hh or Notch target is indicative of inhibition of the Hh or Notch signaling pathway, respectively, in the cell line and indicative of the potential of the candidate as a therapeutic in the treatment of medulloblastoma.
Once obtained, a medulloblastoma-forming clonogenic cell line may be used to prepare a transgenic non-human animal model of medulloblastoma which mimics the disease in humans using conventional methodology. Methods of preparing transgenic animals are well-established in the art. In one embodiment, a method of preparing such an animal model includes injecting an amount of the cell line directly into the cerebella of the animal. Following a sufficient period of time, such as a period of at least about 5 weeks, and preferably a period of greater than 5 weeks, e.g. 8-12 weeks, evidence of medulloblastoma may be observed in the animal including ataxia, weight loss and an increase in intracranial pressure resulting in headaches, sickness (vomiting), sight problems, muscle weakness, fatigue and behavioural changes. Suitable animals for use in this aspect of the present invention may be any non-human mammal, including, without limitation, rodents such as mice, rats, hamsters, and gerbils, rabbits, cats, and dogs.
The non-human medulloblastoma animal model may also be used in a method of screening candidate compounds for therapeutic utility to treat medulloblastoma In particular, as the medulloblastoma animal model comprises the medulloblastoma-forming cells of the invention and these cells retain Hedgehog and Notch signaling activity in vitro, the animal model may be used to identify specific inhibitors that block these important developmental and cancer-associated pathways. A candidate compound may be administered to a medulloblastoma animal model, for example, by injection to the cerebellum or by systemic administration (oral, intravenous, intraperitoneal) before or after a tumor develops. The animal is then monitored for evidence that the candidate compound is inhibiting medulloblastoma formation or is causing medulloblastomas to reduce in size or disappear.
The present invention is described by reference to certain embodiments thereof; however, it will be understood by one of skill in the art that other embodiments of the invention as described and/or defined in the claims may be possible.
References noted herein are incorporated by reference.
Embodiments of the invention are described by reference to the following specific examples which are not to be construed as limiting.
C57/B6 Trp53+/− mice (Jackson Laboratory, Maine, USA) and Ptc1+/− mice were mated to generate a Ptc1+/−p53+/− breeding colony. Mice with symptoms of medulloblastoma, domed appearance of the head, unsteadiness of gait, were sacrificed by cervical dislocation. Brains were removed and tumours microdissected, dissociated by gentle pipetting in PBS and filtered through a 70 cm nylon filter. Cells were grown in serum free conditions with 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (FGF) as previously described (Diamandis, P. et al. Nat Chem Biol 3, 268-73 (2007); Reynolds, B. A. & Weiss, S. Science 255, 1707-10 (1992)). For pharmacological studies, 5000 cells/well were grown in 96 well plates and treated with 5 μM cyclopamine (an inhibitor of Hh signaling) or 10 μM DAPT (an inhibitor of Notch signaling) for a week. 50 μl fresh media and inhibitor was added every second day. For differentiation analysis, cells were grown in DMEM/F12 media supplemented with 10% foetal bovine serum for 7 days. MTT assays were performed as previously described (Diamandis, P. et al. Nat Chem Biol 3, 268-73 (2007))
Freshly dissociated tumour cells were cytospun onto glass slides (105 cells/slide) and established cell lines were grown on glass coverslips. Cells were fixed in −20° C. methanol for 20 min. 4% paraformaldehyde fixed tissue was paraffin embedded and sectioned to generate 61 μm tissue slices. Cells and tissue sections were processed by standard protocol and stained with antibody as indicated in Table 1 below:
Hh and Notch target genes were analyzed by RT-PCR and/or Western blot by standard procedures using the primers in the following Table 2. Where indicated, cells were analyzed after 7 days growth in the absence or presence of 5 μM cyclopamine or 10 μM DAPT.
100,000 cells from established cell lines were suspended in approximately 2 μl cold PBS. NOD/SCID mice were prepared for surgery as previously described (Singh, S. K et al. Nature 432, 396-401 (2004)). Cells were injected into the right hemisphere of the cerebellum using a rodent stereotaxic headframe.
A phenotypic analysis of spontaneous medulloblastomas arising in mice deficient for one Ptc1 allele, alone or in combination with loss of the p53 tumor suppressor protein (Ptc1+/−p53+/+, Ptc1+/−p53+/−, or Ptc1+/−p53−/−), was performed. The expression of multiple neural lineages including MAP2+, NeuN+ and βIII-tubulin+ neurons, GFAP+ astrocytes and Math1+ GCPs was observed. In contrast to these abundant cell types, a subpopulation of Nestin+ and Sox2+ cells were detected, generally in close proximity to the vasculature: the putative CSC niche.
To better characterize the expression pattern of individual Ptc1+/− MB cells, freshly dissociated tumor cells were stained for the markers observed in situ. While 96% of the cells expressed Math1, and a large proportion expressed GFAP or MAP2, a smaller proportion of cells were Nestin+ (
Cells were isolated from Ptc1+/−p53+/+, Ptc1+/−p53+/−, Ptc1+/−p53−/− and irradiated (IR) Ptc1+/− medulloblastomas in vitro were then studied. Twenty-four hours after microdissection, dissociation and plating in serum free conditions (containing EGF and FGF), cells amalgamated into clusters, the majority of which did not go on to proliferate (
The clonogenic frequency of Ptc1+/− medulloblastoma cells was determined by limiting dilution growth analysis (Tropepe, V. et al. Dev Biol 208, 166-88 (1999)) of freshly dissociated tumor cells (
Loss of WT Ptc1 expression is thought to be a major contributing factor to tumor development in these mice. Medulloblastoma cell lines were genotyped, all of which originated from Ptc1+/− mice, and a WT Ptc1 allele and mutant allele (marked by a Neo cassette) was detected in seven of 11 established, long-term cultured cell lines (
Ptc1+/− medulloblastoma harbor activated Hh and Notch signaling in vivo, while serum derived Ptc1+/− medulloblastoma cell lines do not and are thus a poor representation of the disease. The in vitro expression of Hh and Notch target genes in the cells were studied and their response to pharmacological pathway inhibitors were tested. Cell lines with differing Ptc1 and p53 genotypes (n=6) all demonstrated constitutively activated Hh and Notch signaling, expressing all Gli transcripts, Ptc2, Hes1 and Hes5 mRNA (
Finally, to determine the tumourigenic capacity of the Ptc1+/− medulloblastoma cell lines, 105 Nestin+Sox2+ cells were injected into the cerebella of NOD/SCID mice as shown in the schematic (
To determine if mouse medulloblastoma cells recapitulate a CSC hierarchy, an in vivo limiting dilution analysis (Goodrich et al. Science 277, 1109-1113 (1997)) of freshly isolated tumor cells derived from spontaneous medulloblastomas from Ptc1+/− mice was performed alone or in combination with p53 deficiency (Ptc1+/−p53+/+ or Ptc1+/−p53−/−) as set out in Table 3.
Following orthotopic transplantation of cells into the cerebella of NOD/SCID mice, it was determined that injecting ≧5×104 Ptc1+/−p53−/− medulloblastoma cells, but not ≦1×104 cells, consistently generated medulloblastoma in recipient mice.
Fluorescence-activated cell sorting (FACS) of freshly dissociated, uncultured Ptc1+/− medulloblastoma cells for CD15/Lewis X/stage specific embryonic antigen 1 (SSEA-1), a cell surface marker of embryonic and adult mouse neural precursor cells, was then performed. The abilities of CD15+ cells (representing 10-30% of the medulloblastoma cells) and CD15− cells to initiate tumors after orthotopic injection was compared. The results are set out in Table 4 and as illustrated in
In six of seven injections into mice, including 1+/− MB of all p53 genotypes, CD15+ cells were capable of transplanting the disease by 12 weeks, and one of eleven mice injected with equal numbers of CD15− cells showed tumor initiation.
Cerebellar granule cell precursors (CGCPs) are believed to be the cells of origin of medulloblastoma cells, particularly in the Ptc1+/− mouse. Given identification that tumourigenic medulloblastoma cells display similar characteristics to normal neural stem cells, the initiation of medulloblastoma cells from stem cells prior to the birth of Math1+ CGCPs was investigated. Ethyl-N-Nitrosoamine (ENU), a chemical carcinogen known to induce multiple tumor types in rats and mice when administered during development, was administered (25 mg/kg ENU) intraperitoneally to timed-pregnant Ptc+/− mice on e10.5, e14.5 or e17.5. Exposed litters were monitored for up to six months (
Thus, a mouse model of medulloblastoma that retains a stem cell hierarchy and is representative of human medulloblastoma disease is herein provided. The cells capable of expansion demonstrate the stem cell properties of self-renewal and multilineage differentiation. Nestin and Sox2 may be used to identify cells that can be expanded in serum free conditions and initiate the formation of heterogeneous medulloblastoma in allogeneic recipients.
Interestingly, Math1 protein expression did not identify a subpopulation in the primary or secondary tumors in vivo, and was expressed in both undifferentiated and differentiated cells in vitro. Recently, it was reported that Math1+ GCPs from normal cerebella and Ptc1+/− tumors differentiate in response to FGF. In contrast, the present results indicate that Nestin+ and Sox2+ cells are enriched when grown in serum free conditions with EGF and FGF. Further, these stem cell markers define the tumourigenic cells within medulloblastoma.
The present observations also demonstrate the tremendous potential that serum free-derived cancer cell lines provide. EGF and FGF culture conditions allow enrichment and expansion of cells that retain stem cell properties, tumour-initiating capacity, and activated developmental signaling pathways in vitro. The present genetic analysis of Ptc1 genotype demonstrates the resemblance of present cell lines to the primary tumour.
| Number | Date | Country | |
|---|---|---|---|
| 60935960 | Sep 2007 | US |
