Experimental Model for human proliferative Breast Disease
Gerald Hurowitz, M.D.·Published 13 June 2008·8 min read
Key points
Recently, we established a new and unique model of early human breast cancer progression.
MCFlOAneoT is a cell line derived from a normal human breast epithehal hne, which spontaneously immortalized (Soule et al., 1990) and was then transfected with mutated H-ras (Basolo et al., 1991).
The reproducible establishment of representative stages in early breast cancer progression from the MCF1 OAT model offers an unprecedented opportunity to analyze critical events in human breast carcinogenesis.
The MCFIOAT model clearly is a precursor cell line that forms all intermediate stages of prohferative breast disease.
Recently, we established a new and unique model of early human breast cancer progression. This model, which is called "MCFIOAT", consists of preneoplastic human breast epithelial cells that are able to grow in immune-deficient mice where they undergo a sequence of progressive histological changes culminating in cases of frank neoplasia in about 25% of the animals (Miller et al., 1993; Dawson et al., 1996).
MCFlOAneoT is a cell line derived from a normal human breast epithehal hne, which spontaneously immortalized (Soule et al., 1990) and was then transfected with mutated H-ras (Basolo et al., 1991). MCFlOAneoT is a heterogeneous mixture containing a subpopulation that is a multipotent human breast epithelial stem cell that can be selected in vivo by its ability to form persistent lesions in nude/beige mice (Miller et al., 1993). Cell lines established from lesions after serial transplantation into nude/beige mice (MCF10AT# where # represents transplant generation) show more severe atypia at earlier times post-transplantation than does MCFlOAneoT (Dawson et al., 1996). Although ER can not be detected (by Abbot antibody) on cells in xenograft lesions, Dr. P.V.M. Shekhar has shown that the ras- transfected MCFIOAT cells (but not MCFIOA) do express functional ER (Manuscript submitted).
The reproducible establishment of representative stages in early breast cancer progression from the MCF1 OAT model offers an unprecedented opportunity to analyze critical events in human breast carcinogenesis. Normal ducts form initially but become proliferative and sporadically progress to carcinoma in some cases. The ducts formed in xenografts are bilayered, being composed of both myoepithelial and luminal epithelial layers. These normal structures may persist for months in immune-deficient mice.
The MCFIOAT model clearly is a precursor cell line that forms all intermediate stages of prohferative breast disease. Results with our model strengthen the perception of progression from hyperplasia to atypical hyperplasia to invasive breast carcinoma.
We confirmed the invasive phenotype in the MCFIOAT lesions classified as invasive carcinoma by demonstrating the loss of basement membrane ufilizing a silver stain method (Tait et al., 1996a). Unlike the MCFlOAneoT (i.e., ras- transfected cells prior to in vivo selection), all variants derived from xenografts formed intermediate lesions that resemble the lesions seen in human PBD including (PDWA), atypical hyperplasia, and DCIS (Dawson et al., 1996).
It is a widely held point of view that mutations in differentiated epithelial cells are of little importance due to limited growth potential of such cells and that cancer results only from mutations in stem cells. Indeed, we have found that MCFIOAT lines contain stem cells able to differentiate in xenografts into myoepithelial as well as luminal epithelial cells (Tait et al., 1996b). Although ducts formed by the stem cell appear to be normal in most respects, cytokeratin differentiation is abnormal.
Written and revised by
Gerald Hurowitz, M.D.
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MCFIOAT cells express both cytokeratin 14 and cytokeratin 18 (Pauley et al., 1996), a pattern consistent with a stem cell able to generate both cytokeratin 14-expressing myoepithelial cells and cytokeratin 18-expressing luminal cells (Taylor -Papadimitriou et al., 1989). However, neither myoepithelial nor luminal epithelial cells in the xenograft lesions consistentiy express either cytokeratin 14 or 18 (not shown).
MCFIOAT stem cells are unique, differing from previously described human breast and rat mammary stem cells in their capacity to produce organized ductular structures with myoepithelium properly oriented between a basement membrane and the luminal epithelium in situ in xenografts. Although our failure to consistentiy detect cytokeratins 14 and 18 in situ may be due to technical difficulties with the specific antibodies used, the antibodies did stain the appropriate cell layers in control, formalin-fixed, paraffin-embedded human breast tissue. The failure of MCFIOAT cells to differentiate fully in xenografts may be critical to their preneoplastic nature. {pagebreak}
As discussed previously, although ras mutations are rare in breast cancer, over-expression of ras p21 is frequently detected. Mutation of ras is just one of a number of ways that ras signal transduction pathways are activated. The relative infrequency with which ras mutations are seen in human breast cancer may reflect a very large number of active growth factor receptors in breast epithelium which, whether mutant or amplified, result in overexpression of ras. The mechanism(s) by which ras is overexpressed in human breast cancer is not known.
Normal c-Ha-ras-transfected cells, MCFlOAneoN (Basolo et al., 1991), have an extra copy of c-Ha-ras but the protein is not overexpressed (unpublished) and the cells do not form xenograft lesions (Miller et al., 1993). Insertion of the mutant ras resulted in increased p21 protein and transformation of MCFIOA cells. Although the preneoplastic phenotype in xenografts was observed following ras transfection, we have found that not all clones of MCFlOAneoT are able to form lesions in vivo (Miller et al., 1996).
Restriction size fragment analysis demonstrated, however, that clones unable to form preneoplastic lesions retained the activated c-Ha-ras and confirmed that the insertion site of the activated c-Ha-ras was the same for these clones as for the variants selected for ability to form lesions in vivo. Furthermore, Western blotting with antibodies specific for the codon 12 valine c-Ha-ras demonstrated that mutant p21 protein was comparable in nonlesion-forming clones and lesion-forming variants as well (Miller et al., 1996).
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Thus, activated c-Ha-ras is not sufficient to produce the preneoplastic phenotype of the MCFl OAT human breast stem cells. This was an important finding because it offers the opportunity to identify the genetic alteration(s) involved in this critical early stage in proliferative breast disease by comparing the nonlesion-forming clones with the preneoplastic variants selected by their ability to form preneoplastic lesions.
The karyotype of the MCFIOAT cells in vivo is remarkably stable despite longterm passage in xenografts. The only alterations have occurred during extended in v/rro maintenance. An additional marker chromosome, t(3;17)(pl3;pl2), appeared in MCFIOA cells in vitro between the time of immortalization and the time of transfection with T24 ras.
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Likewise, MCFlOAneoT cells were initially identical to the untransfected MCFIOA cells but acquired an additional, apparently normal copy of chromosome 9 prior to the first xenograft experiment. Subsequent MCF10AT# variants isolated from xenograft lesions have been unchanged regardless of the histology of the lesion from which the cells were derived.
However, we do not suggest that progression to carcinoma occurs without karyotypic alterations in the MCFIOAT model. Breast cancer cells are notoriously difficult to establish in culture from solid tumors; therefore we hypothesize that the preneoplastic stem cell with the depicted karyotype has a growth advantage in vitro. {pagebreak}
Thus, even in xenografts that appear to be pure carcinoma, the precursor stem cell exists covertly and grows when primary cultures are established in vitro. The fact that these variants established from xenografts have been consistently preneoplastic stem cells rather than carcinomas supports this concept. When clones are established directly from xenografts by modifying the tissue disaggregation method, other karyotypes have been isolated.
Thus, we believe that cells with other karyotypes do develop within the lesions in vivo but are overgrown by the dominant stem cell in vitro. We have recently isolated tumorigenic variants (i.e., carcinomas form directly with no precursor lesion) by serially transplanting lesions by trocar before establishing cells in culture.
Although the MCFIOAT xenografts progress to carcinoma with approximately a 25% incidence, rapidly growing xenografts are rare. Carcinoma has generally been diagnosed only after removal of the lesion and histologic examination. Indeed, the largest lesion to date has weighed 200 mg and most weigh less than 50 mg. When one considers the natural history of human breast cancer, which takes years to develop, the slow growth of these relatively early carcinomas is not surprising.
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However, results with the MIB-1 antibody against the Ki-67 nuclear proliferation antigen clearly demonstrate that the proUferative rate increases with progression in the MCFIOAT xenograft model from 5% in simple hyperplastic lesions to 10% in CIS and 20% in invasive cancers. These rates resemble those reported for lesions from human patients (Barbareschi et al., 1992; PaveUc et al., 1992; Jensen, V. et al., 1995).
The MCFIOAT model should be valuable to analyze genetic alterations in the development of breast cancer. Specific genes can be experimentally altered in the cells to determine the effect on the incidence of cancer in xenografts, and new genes may be identified by comparing cells before and after spontaneous progression in xenografts.
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A number of genes implicated in breast cancer are being analyzed in the MCFIOAT system. Immunohistochemical studies with DOT antibody to p53 (detects both wild type and mutant forms) found that staining was not a feature of hyperplastic lesions, CIS, or adenocarcinomas developing in MCFIOAT xenografts; significant increased expression of p53 was detected only in squamous carcinomas (Iravani et al., 1996). A number of investigators have found that/753 is not mutated in MCFIOA (Diella et al., 1993; Gudas et al., 1995; Merlo et al., 1995) nor MCFIOAT cells that form preneoplastic lesions in xenografts (Chen, Y.Q. et al.. 1995; Shekhar et al., 1995).
Taken together, these data suggest that mutated p53 does not play a significant role in producing the preneoplastic phenotype or in progression to adenocarcinoma but may be important in generation of squamous carcinoma. However, the expression of a conformationally altered, but not mutated, p53 does increase with successive transplant generations (MCF10AT1 / MCF10AT2B/ MCF10AT3B) and is accompanied by diminished normal/755 function (Shekhar et al., 1995).
Although erbB-2 is rarely amplified in hyperplastic human biopsy specimens, erbB-2 overexpression can be detected beginning at the stage of atypical hyperplasia in the MCFIOAT xenograft model (Iravani et al., 1996). As in human cancer, the proportion of erbB-2 - overexpressing MCFIOAT lesions increases at the stage of CIS and decreases somewhat in invasive adenocarcinomas. Although erbB-2-expression increases two- to threefold with in vivo passage (Wang and Miller, unpublished), MCFlOAneoT cells transfected with erbB-2 (MCFIOHE) are not tumorigenic (Ciardiello et al., 1992), nor do they form persistent lesions in immune-deficient mice (Miller, unpublished).
As discussed previously, because clones of MCFlOAneoT may not form persistent preneoplastic xenograft lesions, we hypothesize that a defect in addition to the T24 ras is necessary to produce the preneoplastic phenotype observed in MCFlOAneoT and MCFIOAT variants. We suggest that the MCFIOHE cells, which were cloned from MCFlOAneoT after transfection with the erbB-2 expression construct (Ciardiello et al., 1992), lack that hypothetical genetic defect. Growth in vivo selects for the second genetic defect and clones of MCFIOAT variants do form lesions.
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