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[Cancer Research 64, 857-863, February 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Polyclonal Development of Mouse Mammary Preneoplastic Nodules

William C. Kisseberth1 and Eric P. Sandgren2

1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, and 2 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies of cellular interactions are critical to the understanding of tumorigenesis. Although many studies have demonstrated a monoclonal composition of advanced neoplasms in humans and mice, the clonal composition of smaller, antecedent lesions has been studied less thoroughly. To examine the clonal development of breast cancer, we generated chimeric mammary glands using mouse mammary epithelium with an inherited predisposition for neoplasia. Analysis of whey acidic protein-transforming growth factor-{alpha} transgenic mouse mammary glands, chimeric for two different cell lineage markers, revealed that mammary ducts and alveoli are polyclonal, and putative early preneoplastic lesions, hyperplastic alveolar nodules (HANs), frequently are polyclonal. Furthermore, the chimeric patch patterns in individual HANs were similar to the patterns observed in pregnant chimeric mammary glands. Thus, polyclonality in HANs appears to reflect persistence of the polyclonal architecture of ducts and/or alveoli, suggesting that hyperplasia formation can be the result of non-cell autonomous local tissue microenvironmental influences on groups of cells, rather than clonal progression of a single initiated cell.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The multistage model of carcinogenesis emphasizes somatic mutation as the neoplastic initiating event, which then is followed by accumulation of additional genetic and epigenetic changes in the initiated cell or its progeny. Collectively, these events lead to the emergence of a clonal dominant cell population. This model predicts that the earliest lesions in this process will be monoclonal. Until recently, most evidence has supported a monoclonal origin for solid tumors, including tumors of the colon, skin, and breast in humans (1, 2, 3, 4, 5, 6, 7, 8) and in carcinogen-treated rodents (9, 10, 11) . However, studies have demonstrated polyclonality in at least some early preneoplastic lesions and epithelial tumors (12, 13, 14) .

Although most analyses of animal and human tumors support a monoclonal lesion composition, interpretation of these studies is complicated by two related problems:

(a) When addressing the question of tumor clonality, it is important to differentiate between clonal origin and clonal evolution. A clonal origin would follow from the emergence of a single cell with a growth advantage that clonally expands into a neoplasm. In contrast, clonal evolution implies a later event that is the result of selective growth of a subpopulation of tumor cells (15 , 16) . Monoclonality at a late stage of tumor progression thus may not reflect the cellular composition of the originating neoplastic cell population. Similarly, polyclonality at this late stage is difficult to interpret. Mammary neoplasms tend to be cytogenetically complex, with individual cells often having different, seemingly unrelated karyotypes (17 , 18) . This diversity may reflect the underlying genetic instability within advanced neoplasms rather than the original cellular composition. For these reasons, it is important to analyze the smallest recognized lesions, which are more likely to represent the initially transformed cell population.

(b) Most methodologies used to investigate clonality, especially in human tumors, involve biochemical or molecular evaluation of homogenized tissue. These techniques include analysis of patterns of inactivation of polymorphic X chromosome-linked genes such as glucose-6-phosphate dehydrogenase (19) , glycerophosphate kinase, the androgen receptor gene (HUMARA; Ref. 2 ), and hypoxanthine phosphoribosyltransferase (20) ; detection of somatic mutations (21 , 22) ; studies of viral integration site by Southern blot analysis (23) ; and detection of microsatellite instability (24) . Using these methods, the genotype of the entire tissue sample is identified without determining the genotype for individual cells. The sensitivity of these procedures can be relatively low. For example, for X-inactivation studies using homogenized tissue, a monoclonal cell population must comprise at least 20% of the sample population to be recognized reliably (25) . Conversely, a polyclonal composition may be inferred mistakenly if nonneoplastic tissue contamination (e.g., stroma) is too great.

Evaluation of the clonal origin of breast cancer has yielded conflicting answers. Human breast neoplasms and preneoplastic proliferative lesions occasionally show cytogenetic abnormalities that suggest the presence of multiple clones (26) . Recently, Going et al. (14) used a combined approach evaluating X-inactivation and androgen receptor exon I CAG polymorphism to propose that one-third of human mammary carcinomas analyzed were polyclonal. Nevertheless, other studies indicate that some of the same lesions appeared to be clonal (7 , 27, 28, 29) . Experimentally, mouse mammary tumor virus-induced preneoplastic hyperplastic alveolar nodules (HANs) and tumors have been suggested to be monoclonal, based on restriction fragment length polymorphism analysis of proviral insertion sites (30) . Also, in rats, methylnitrosourea-induced mammary carcinomas possessed monoclonal mutations in the ras oncogene (11) .

Whey acidic protein-transforming growth factor-{alpha} (WAP-TGF{alpha}) transgenic mice overexpress mature, soluble TGF{alpha} in mammary epithelium (31) . Female mice that overexpress TGF{alpha} in mammary epithelium develop mammary carcinomas, temporally preceded by formation of HANs in the mammary gland (32, 33, 34) . HANs histologically resemble lactating epithelium in a nonlactating gland. HANs are serially transplantable (in some cases indefinitely), display increased neoplastic potential compared with normal mammary epithelium, and also can be induced by mouse mammary tumor virus, hormones, and chemical carcinogens in rodents (35 , 36) . For these reasons, HANs have been considered to represent a preneoplastic mammary epithelial cell population. Here we describe studies of transplantation chimeric mammary glands in which each gland is composed of two uniquely marked epithelial cell populations that both carry the same TGF{alpha} transgene. We have used this cell marking system to determine the clonal composition of normal epithelium and of TGF{alpha}-induced epithelial hyperplasias (HANs) in the mouse mammary gland.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mouse Strains.
The 804-2 line of R26-hPAP transgenic mice, which expresses the marker gene human placental alkaline phosphatase (hPAP) in all tissues (37) , and the K18TG2 line of transgenic mice, which expresses human cytokeratin 18 (hCK18) in simple epithelia, including mammary epithelia (38 , 39) , were generated and maintained on the FVB/N background. The 3573-2 line of WAP-TGF{alpha} transgenic mice, [TgN(WAPTgfa)215Bri], was described previously (31) . WAP-TGF{alpha} mice originally were generated in a C57BL/6 x SJL background, but for this study were used after >=10 generations of backcrossing into the FVB/N strain. The hCK18 mice were provided by Robert Oshima (The Burnham Institute, La Jolla, CA). All mice were housed in Association for Assesment and Accreditation of Laboratory Animal Care accredited facilities and handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Experimental procedures were approved by the University of Wisconsin-Madison Animal Care and Use Committee.

Genotyping.
Transgenic mice were identified by PCR analysis using the following primer pairs: R26-hPAP, SVpoly-1f (5'-CTGATGAATGGGAGCAGTGGTGGAATG-3') and SVpoly-2r (5'-GCAGACACTCTATGCCTGTGTGGAG-3'); hCK18, hCK18–5f (5'-GCATGAGCAACCACGCCCAGCCTA-3') and hCK18–6r (5'-CTGCTGAAGGCTGGGAGGCAAAC-3'); and WAP-TGF{alpha}, TGF-PC3f (5'-TGTCAGGCTCTGGAGAACAGC-3') and TGF-E4r (5'-CACAGCGAACACCCACGTACC-3'). Twenty-five µl of reaction mixture containing genomic DNA from tail were subjected to the following conditions for R26-hPAP and WAP-TGF{alpha} genotyping: (a) 92°C for 110 s; (b) 35 cycles of: 45 s at 92°C, 60 s at 60°C, and 60 s at 72°C; and (c) 72°C for 5 min. For hCK18 transgene genotyping, genomic DNA was subjected to the following conditions: (a) 92°C for 120 s; (b) 34 cycles of: 60 s at 92°C, 90 s at 60°C, and 110 s at 72°C; and (c) 72°C for 7 min. R26-hPAP mice alternatively were genotyped by enzyme histochemistry performed on peripheral blood (37) .

Generation of Mammary Gland Chimeras.
Chimeric mammary glands were produced by transplantation of 5 x 104 collagenase/trypsin-isolated mammary epithelial cells from each of two genotypes into cleared mammary fat pads of 3-week-old syngeneic FVB/N nontransgenic female recipients (Refs. 40 , 41 ; Fig. 1Citation ). Four different genotypes in two combinations were used: bitransgenic R26-hPAP/WAP-TGF{alpha} and bitransgenic hCK18/WAP-TGF{alpha} cotransplanted, or marker-only R26-hPAP and hCK18 cotransplanted. Recipient mammary fat pads were "cleared" of host epithelium by surgical excision of the mammary rudiment before it extended into the fat pad. Transplanted mammary epithelial cells divide and differentiate to produce a morphologically normal mammary gland. We used this procedure to create chimeric mammary glands in which each mammary epithelial cell contains: (a) the WAP-TGF{alpha} transgene and one of two marker transgenes (R26-hPAP/WAP-TGF{alpha} {leftrightarrow} hCK18/WAP-TGF{alpha}); or (b) one of two marker transgenes only. For subsequent analyses, chimeric or control mammary glands were collected from virgin, day 18 pregnant, or nonpregnant multiparous females. Mice were not mated until at least 8 weeks after epithelial cell transplantation.



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  		Fig. 1. Generation of transplantation chimeras. Mammary epithelial cells from WAP-TGF{alpha} transgenic mice are genetically marked by crossing WAP-TGF{alpha} mice with mice expressing a marker transgene (R26-hPAP or hCK18). Equal numbers of collagenase/trypsin-isolated adult mammary epithelial cells from R26-hPAP/WAP-TGF{alpha} and hCK18/WAP-TGF{alpha} bitransgenic mice were injected into cleared mammary fat pads of syngeneic recipient female mice, 3 weeks of age. After transplantation, they reconstitute the fat pad with chimeric mammary ductal and alveolar structures. Epithelial cells derived from transplanted hCK18 transgene-marked cells are identified using immunohistochemistry with an anti-hCK18 monoclonal antibody (brown cells). R26-hPAP transgene-marked cells do not stain with this procedure and are visualized with a hematoxylin counterstain (blue cells) or by specific staining for hPAP.

 
hCK18 Immunohistochemistry and hPAP Histochemistry.
Reconstituted mammary fat pads or tumors were fixed in 4% paraformaldehyde at 4°C for 4 h. Fixed tissues were dehydrated through graded alcohols to Hemo-De (Fisher Scientific, Pittsburgh, PA) and paraffin embedded, then 5-µm sections were cut and mounted on poly-L-lysine-coated slides. hPAP histochemistry was performed as described previously (37) . Sections were dewaxed in Hemo-De, rehydrated, heated at 65°C in substrate buffer [0.1 M Tris-HCl (pH 9.5), 0.1 M NaCl, and 5 mM MgCl2] for 30 min to block endogenous alkaline phosphatase activity, and then incubated with fresh buffer containing 0.17 mg/ml of the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Sigma, St. Louis, MO). Samples were incubated 18–48 h at 37°C, rinsed, dehydrated through graded alcohols to Hemo-De, and mounted under glass coverslips. Immunohistochemistry for human cytokeratin 18 using mouse monoclonal antibody (clone DC10; BioGenex, San Ramon, CA) at a dilution of 1:10–1:20 was used in a standard peroxidase-based protocol. Briefly, 5-µm tissue sections were dewaxed and rehydrated through graded alcohols. Antigen retrieval was performed by microwaving the slides in 6 M urea (pH 6.0) for 10 min at 50% power in a 1450-W microwave. Endogenous peroxidase activity was quenched by incubation of the slides in 0.3% hydrogen peroxide in methanol for 20 min on ice. Immunohistochemistry then was performed with the Supersensitive peroxidase system according to the manufacturer’s directions (BioGenex). Sections were developed in diaminobenzidine substrate (Sigma). Sections were counterstained with Mayer’s hematoxylin for 2 min. Sections that were double-labeled first were stained with hPAP histochemistry with 48-h substrate incubation, followed by hCK18 immunohistochemistry.

Determining Number of Chimeric HANs in Reconstituted Mammary Glands.
The number and clonal composition of HANs was determined in a single random hCK18 immunostained and hematoxylin counterstained sagittal section from each reconstituted gland. Individual anatomical structures (mammary ducts, alveoli) in the entire stained tissue section were analyzed for the presence of all brown (hCK18+), all nonbrown (hCK18-), or mixed brown/nonbrown (hCK18+/hPAP-) cells. HANs present within the glands were analyzed similarly. HANs were defined as persistent alveolar structures containing three or more alveolar lumenal profiles in a nonpregnant, nonlactating mammary gland. Individual HAN profiles were completely surrounded by adipose tissue or had a single point of contact with another HAN but remained separated by a discrete layer of denser stromal tissue. Selected glands were stained histochemically with BCIP for hPAP expression to confirm that hCK18-negative cells carried the hPAP marker transgene. In addition, the level of chimerism within HANs was characterized further as being at the cell level (hCK18+ and hCK18- cells present in the same alveolar cross-section), alveolar level (uniformly hCK18+ or hCK18- alveoli present in the same HAN), or both (chimerism present at both the cell and alveolar level in the same HAN).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
hCK18 and R26-hPAP as Cell Lineage Markers in Mammary Gland.
We used the R26-hPAP and hCK18 transgenic lines as cell lineage markers in chimeric mice. R26-hPAP transgenic mice express human placental alkaline phosphatase in all tissues and have been used to mark transplanted cells in previous studies (37 , 42 , 43) . hCK18 transgenic mice express human cytokeratin 18 under the control of its own regulatory elements (38 , 39) . The monoclonal antihuman cytokeratin 18 antibody that we used demonstrated strong immunoreactivity in alveolar mammary epithelia of hCK18 transgenic mice but did not recognize the mouse cytokeratin 18 homolog, endoB, in nontransgenic mice. Ductal mammary epithelial cells also stained strongly in hCK18 transgenic mice but did not stain or stained very weakly in nontransgenic mice (data not shown). To determine whether R26-hPAP and hCK18 are expressed in transplanted WAP-TGF{alpha} mammary epithelium, we crossed WAP-TGF{alpha} transgenic mice with R26-hPAP or hCK18 marker mice and transplanted isolated mammary epithelial cells into cleared mammary fat pads of nontransgenic recipient female mice, 3 weeks of age, as described in "Materials and Methods." Glands from virgin, pregnant, or nonpregnant multiparous females were removed at selected times, fixed, sectioned, and stained to localize the hCK18 and/or hPAP markers. Reconstituted mammary glands from WAP-TGF{alpha}/hCK18 single-marker control recipient mice showed strong and spatially consistent hCK18 immunostaining in all epithelial cells and HANs (Fig. 2, A and C)Citation . hPAP staining of reconstituted mammary glands from WAP-TGF{alpha}/R26-hPAP single-marker control recipient mice showed hPAP expression of variable intensity in epithelial cells and HANs (Fig. 2, B and D)Citation . The strength of hPAP staining varied within an individual gland and between glands. We also observed hPAP histochemical staining of the nontransgenic stroma (adipocytes) immediately adjacent to R26-hPAP mammary epithelium in reconstituted glands. We concluded that in this system, the hCK18 transgenic lineage was the more precise and consistent marker strain. Therefore, we performed our initial analyses on chimeric mammary glands using hCK18 immunohistochemistry only and then in selected cases confirmed that the hCK18-negative epithelium was marked by the R26-hPAP transgene.



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  		Fig. 2. Mammary gland development in transplantation chimeric mammary glands. Reconstituted WAP-TGF{alpha}/R26-hPAP, WAP-TGF{alpha}/hCK18, and WAP-TGF{alpha}/R26-hPAP{leftrightarrow} WAP-TGF{alpha}/hCK18 mammary fat pads were removed from recipient mice and then fixed and stained as described in "Materials and Methods." A, day 18 pregnant WAP-TGF{alpha}/hCK18 reconstituted mammary gland. All lumenal mammary epithelial cells are hCK18-positive. B, day 18 pregnant WAP-TGF{alpha}/R26-hPAP reconstituted mammary gland. There is variable hPAP staining intensity of mammary epithelium and light nonspecific staining of adjacent nontransgenic recipient stroma. C, all HANs in this WAP-TGF{alpha}/hCK18 reconstituted mammary gland are composed of only hCK18-positive epithelial cells. D, all HANs in this WAP-TGF{alpha}/R26-hPAP reconstituted mammary gland are composed of only hPAP-positive mammary epithelial cells. E, virgin WAP-TGF{alpha}/R26-hPAP{leftrightarrow} WAP-TGF{alpha}/hCK18 mammary gland. Some mammary ducts are polyclonal. F, day 18 pregnant WAP-TGF{alpha}/R26-hPAP{leftrightarrow} WAP-TGF{alpha}/hCK18 mammary gland. Some alveoli are polyclonal and have a variable genotype marker composition. G, day 18 pregnant WAP-TGF{alpha}/R26-hPAP{leftrightarrow} WAP-TGF{alpha}/hCK18 mammary gland. Note the variable patch patterns. Some portions of the gland are composed of only hCK18-positive or hCK18-negative epithelial cells, and other areas are composed of mixtures of both. hCK18-negative epithelial cells stain blue with BCIP, confirming their R26-hPAP origin. H, day 18 pregnant WAP-TGF{alpha}/R26-hPAP{leftrightarrow} WAP-TGF{alpha}/hCK18 mammary gland. hCK18-negative epithelial cells stain blue with BCIP. A–D and H, x100; E and F, x200; G, x40.

 
Generation and Characterization of Chimeric Mammary Epithelium.
To determine the clonal composition of mammary ducts and alveoli in reconstituted mammary glands, hCK18 immunostained R26-hPAP/WAP-TGF{alpha} {leftrightarrow} hCK18/WAP-TGF{alpha} chimeric mammary glands from day 18 pregnant and virgin mice were prepared as described above for single-marker control glands and then examined microscopically. The predominant staining pattern observed consisted of relatively large brown or nonbrown patches with variable mixing of the two lineages (Fig. 2, E and F)Citation . For example, on a single tissue section, the following alveolar staining patterns could be identified: (a) all brown cells except for one nonbrown cell; (b) one-half of the alveolus brown and the opposite half nonbrown; and (c) variable numbers and patterns of brown cells alternating with variable numbers of nonbrown cells (Fig. 2F)Citation . Chimeric mammary ducts displayed similarly variable patterns of genotype mixing (Fig. 2E)Citation . Several hCK18-immunostained tissue sections were costained with hPAP histochemistry (Fig. 2, G and H)Citation , demonstrating that nonbrown cells stained blue, indicative of R26-hPAP marker transgene expression.

Histological Examination of Hyperplastic Alveolar Nodules.
To assess the clonal composition of HANs, we examined immunohistochemically stained sections from nonpregnant, parous WAP-TGF{alpha}/hCK18{leftrightarrow} WAP-TGF{alpha}/R26-hPAP chimeric mammary glands. We identified seven reconstituted glands that were informative, containing both hCK18-positive and hCK18-negative epithelial cells and multiple HANs (Table 1Citation and Fig. 3Citation ). Individual HANs from these glands were scored as to whether they were: (a) all hCK18 negative; (b) all hCK18 positive; (c) chimeric at the cell level (i.e., within a single alveolar cross-section, some cells were hCK18 positive and some were hCK18 negative); (d) chimeric at the alveolar level (i.e., some alveolar cross-sections were all hCK18 positive, and some alveolar cross-sections were all hCK18 negative); or (e) chimeric at both the cell and alveolar level. From a total of 134 HANs in these seven chimeric mammary glands, 62 were all hCK18 positive (Fig. 3B)Citation , 20 were all hCK18 negative (Fig. 3C)Citation , and 52 were mixed hCK18 positive/hCK18 negative (39%; Table 1Citation ). Among mixed HANs, 37 were chimeric at the cell level (Fig. 3E)Citation , 2 were chimeric at the alveolar level, and 13 were chimeric at both the cell and alveolar levels (Fig. 3, D and F)Citation . Finally, to determine if the occurrence of chimerism was influenced by HAN size, we counted the number of alveolar lumens (an indicator of relative HAN size) for each chimeric HAN described in Table 1Citation . Chimerism was observed in HANs of all sizes (Table 2)Citation .


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  		Table 1 HAN clonality within mammary glands of WAP-TGF{alpha}/hPAP {leftrightarrow} WAP-TGF{alpha}/hCK18 transplantation chimeras

 


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  		Fig. 3. HAN histology in chimeric mammary glands. Reconstituted WAP-TGF{alpha}/R26-hPAP{leftrightarrow} WAP-TGF{alpha}/hCK18 mammary fat pads were removed from recipient mice and then fixed and stained as described in "Materials and Methods." A, low-power magnification of the mammary gland from chimera 124 after hCK18 immunohistochemistry, showing mammary ducts and HANs containing both hCK18-positive and hCK18-negative components. x40. B, pure hCK18-positive HAN. C, pure hCK18-negative HAN. D, mixed HAN, chimeric at both the cell and alveolar levels. E, mixed HAN, chimeric at the cell level. F, chimera 132, combined hCK18 immunohistochemistry and BCIP histochemistry confirms that hCK18-negative epithelial cells are R26-hPAP-derived. B–F, x200.

 

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  		Table 2 Number of alveoli in chimeric HANs of WAP-TGF{alpha}/hPAP {leftrightarrow} WAP-TGF{alpha}/hCK18 transplantation chimeras

 
Because this analysis is based on staining of a single histological section from each chimeric gland, a portion of any HAN that is out of the plane of section may have been chimeric, even if the examined cross-section appeared monoclonal. Furthermore, HANs containing a pure population of hCK18-positive or hCK18-negative cells may be polyclonal, but this could not be ascertained if the different clones expressed the same marker. Therefore, this experimental approach underestimates the true frequency of polyclonal HANs.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data indicate that many epithelial hyperplasias in WAP-TGF{alpha} transgenic mouse mammary glands are polyclonal. We also show that normal mammary ducts and alveoli generated by mammary epithelial cell transplantation are polyclonal. Furthermore, we show that similar patterns of marker (genotype) mixing can be observed in pregnant and hyperplastic alveoli. These findings suggest that HAN polyclonality reflects persistence of the polyclonal architecture of ducts and alveoli that exists in recipient mice before the onset of disease. This outcome indicates that TGF{alpha}-induced HAN formation can involve the coordinated growth of groups of cells within the context of the local mammary environment, rather than being exclusively a monoclonal event initiated by a single cell with altered growth potential.

Our findings are consistent with the observations of Mintz and Slemmer (44) . In that early study of mouse mammary tumor virus-induced HANs in chimeric mice, the genotype composition of each HAN was inferred from its transplantability into recipients of both chimera donor strains. However, Cardiff et al. (30) reached a different conclusion when they observed monoclonality in hyperplastic outgrowths produced from transplantation of single mouse mammary tumor virus-induced HANs into gland-free fat pads. Because the latter analysis was not performed directly on the HANs but rather on their descendent outgrowths, the possibility of emergence of a clonal dominant outgrowth from an originally polyclonal HAN must be considered.

Our observation of polyclonal mammary ducts and alveoli in transplantation chimeras is consistent with the reported polyclonal composition of the mammary gland in normal mice, based on in situ histochemical detection of polymorphisms in the enzyme glucose-6-phosphate dehydrogenase (45) . The genotype-specific staining patterns of mouse mammary glands in that study and ours suggest that alveoli can originate from more than one cell; however, this may not be true for human breast development. Tsai et al. (46) used X chromosome inactivation analysis and laser capture microdissection of isolated epithelium and concluded that individual lobules and ducts in normal human breast are monoclonal. Recently, Novelli et al. (47) determined that in human breast the terminal ducts and lobules are monophenotypic based on in situ glucose-6-phosphate dehydrogenase histochemistry. However, they concluded that the large X-inactivation patch size confounded assessment of clonality.

The primary evidence suggesting polyclonality of both premalignant human breast lesions and carcinomas has been cytogenetic (17 , 18 , 26 , 48 , 49) , with almost 50% of breast carcinomas and benign breast tumors containing multiple clones lacking common cytogenetic abnormalities (17 , 26 , 49) . However, whether such divergent clones represent a polyclonal origin versus polyclonal evolution after the neoplastic phenotype was established is undetermined (18) .

Studies in other tissues also have suggested a polyclonal development for some epithelial neoplasms. Novelli et al. (50) showed that the majority of the adenomas in a human XO/XY mosaic familial adenomatous polyposis (FAP) patient were polyclonal. Polyclonal adenomas also were found in chimeric Min mice in which both components of the chimera carried the Min mutation and one component also carried a lacZ marker, allowing the clonality of the resulting intestinal adenomas to be assessed (12) . Min mice and familial adenomatous polyposis patients both carry mutations in the adenomatous polyposis coli gene. Using a similar experimental approach using mouse embryo aggregation chimeras expressing different H2 haplotypes, Winton et al. (13) showed that 20% of chemically induced skin papillomas and antecedent foci of epidermal hyperplasia were polyclonal.

Merritt et al. (12) speculated on the origin of mixed intestinal adenomas. Possible explanations for mixed lesions in chimeric mouse tissues include: random collision between lesions, somatic mosaicism for the marker, epigenetic silencing of marker expression, and interaction between multiple clones. Similar to those workers, we have found evidence against the first three of these hypotheses: (a) chimeric HANs were found in fat pads with low HAN multiplicity, arguing against random collision events; and (b) chimeric alveoli never were found in nonpregnant or pregnant hCK18 or R26-hPAP transgenic mice or mammary glands reconstituted with only WAP-TGF{alpha}/hPAP or WAP-TGF{alpha}/hCK18 cells. These observations suggest that somatic mosaicism or epigenetic silencing of markers is not the cause of chimerism, because marker gene expression persisted in all cells in these mice. We believe that the presence of chimeric HANs suggests that they arise via interaction between multiple clones within ductal and alveolar units under the influence of the local microenvironment.

Overall, our findings have significance regarding fundamental mechanisms underlying mammary carcinogenesis:

(a) The presence of polyclonality in small, putatively preneoplastic lesions conflicts with the dogma of a cell autonomous origin for neoplasia, in which a neoplasm originates via a series of changes initiated in a single cell. Specifically, these data suggest that focal morphological changes with an increased neoplastic potential can precede the clonal expansion of a single initiated cell. The polyclonal HAN then represents a heterogeneous "target" cell population within which a clonal tumor might arise. In this sense, our data also call into question how we define preneoplasia. Previously, focal lesions of many different tissues were considered neoplastic if it could be proven that they were clonal. Conversely, polyclonality was considered evidence of a nonneoplastic process.

(b) The development of polyclonal preneoplastic lesions supports clonal evolution, rather than only clonal origin, as an important feature of mammary tumor progression.

(c) Our findings emphasize the importance of non-cell autonomous interactions during mammary tumor progression and complement recent suggestions that interactions between stromal and epithelial cells may provide critical signals that accompany development of neoplasias.

(d) The suggestion that preneoplastic mammary lesions are polyclonal may be important to understanding the potential behavior of individual premalignant breast lesions in women and to developing rational medical strategies for their management. By identifying the factors regulating multicellular interactions in these early lesions, we may find ways to halt their progression to invasive cancer.


   ACKNOWLEDGMENTS
 
We thank Robert Oshima for providing hCK18 transgenic mice and William Dove and Michael Gould for helpful discussions. We also thank Teresa Rose-Hellekant and Meg Bowden for technical assistance.


   FOOTNOTES
 
Grant support: Grant RR00115 (to W. C. Kisseberth) from the NIH.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: William C. Kisseberth, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.

Received 8/28/03. Revised 11/ 7/03. Accepted 11/24/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Vogelstein B., Fearon E. R., Hamilton S. R., Feinberg A. P. Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors. Science (Wash. DC), 227: 642-645, 1985.[Abstract/Free Full Text]
  2. Vogelstein B., Fearon E. R., Hamilton S. R., Preisinger A. C., Willard H. F., Michelson A. M., Riggs A. D., Orkin S. H. Clonal analysis using recombinant DNA probes from the X-chromosome. Cancer Res., 47: 4806-4813, 1987.[Abstract/Free Full Text]
  3. Fearon E. R., Hamilton S. R., Vogelstein B. Clonal analysis of human colorectal tumors. Science (Wash. DC), 238: 193-197, 1987.[Abstract/Free Full Text]
  4. Harada M., Suzuki M., Ikeda T., Kaneko T., Harada S., Fukayama M. Clonality in nevocellular nevus and melanoma: an expression-based clonality analysis at the X-linked genes by polymerase chain reaction. J. Investig. Dermatol., 109: 656-660, 1997.[CrossRef][Medline]
  5. Robinson W. A., Lemon M., Elefanty A., Harrison-Smith M., Markham N., Norris D. Human acquired naevi are clonal. Melanoma Res., 8: 499-5031, 1998.[Medline]
  6. Koyama N., Nishihira J., Nakabayashi H., Nishi S., Hoshi N., Fujimoto S., Shimizu M., Yoshiki T., Sugihara T. Aneuploidy of sex chromosomes in basal cell carcinoma: its clonality and involvement in the development of carcinogenesis. Int. J. Oncol., 16: 15-23, 2000.[Medline]
  7. Noguchi S., Aihara T., Koyama H., Motomura K., Inaji H., Imaoka S. Clonal analysis of benign and malignant human breast tumors by means of polymerase chain reaction. Cancer Lett., 90: 57-63, 1995.[CrossRef][Medline]
  8. Noguchi S., Aihara T., Koyama H., Motomura K., Inaji H., Imaoka S. Discrimination between multicentric and multifocal carcinomas of the breast through clonal analysis. Cancer (Phila.), 74: 872-877, 1994.
  9. Ponder B. A., Wilkinson M. M. Direct examination of the clonality of carcinogen-induced colonic epithelial dysplasia in chimeric mice. J. Natl. Cancer Inst., 77: 967-976, 1986.
  10. Deamant F. D., Iannaccone P. M. Clonal origin of chemically induced papillomas: separate analysis of epidermal and dermal components. J. Cell Sci., 88 (Pt 3): 305-312, 1987.
  11. Lu J., Jiang C., Mitrenga T., Cutter G., Thompson H. J. Pathogenic characterization of 1-methyl-1-nitrosourea-induced mammary carcinomas in the rat. Carcinogenesis (Lond.), 19: 223-227, 1998.[Abstract/Free Full Text]
  12. Merritt A. J., Gould K. A., Dove W. F. Polyclonal structure of intestinal adenomas in ApcMin/+ mice with concomitant loss of Apc+ from all tumor lineages. Proc. Natl. Acad. Sci. USA, 94: 13927-13931, 1997.[Abstract/Free Full Text]
  13. Winton D. J., Blount M. A., Ponder B. A. Polyclonal origin of mouse skin papillomas. Br. J. Cancer, 60: 59-63, 1989.[Medline]
  14. Going J. J., Hanan M., El-Monem A., Craft J. A. Clonal origins of human breast cancer. J. Pathol., 194: 406-412, 2001.[Medline]
  15. Nowell P. C. The clonal evolution of tumor cell populations. Science (Wash. DC), 194: 23-28, 1976.[Abstract/Free Full Text]
  16. Kern S. E. Clonality: more than just a tumor-progression model. J. Natl. Cancer Inst. (Bethesda), 85: 1020-1021, 1993.[Free Full Text]
  17. Teixeira M. R., Pandis N., Bardi G., Andersen J. A., Mandahl N., Mitelman F., Heim S. Cytogenetic analysis of multifocal breast carcinomas: detection of karyotypically unrelated clones as well as clonal similarities between tumour foci. Br. J. Cancer, 70: 922-927, 1994.[Medline]
  18. Heim S., Teixeira M. A. R., Pandis N. Are some breast carcinomas polyclonal in origin?. J. Pathol., 194: 395-397, 2001.[CrossRef][Medline]
  19. Fialkow P. J. Glucose-6-phosphate dehydrogenase (G-6-PD) markers in Burkitt lymphoma and other malignancies. Haematol. Blood Transfus., 20: 297-305, 1977.[Medline]
  20. Sternlicht M., Mirell C., Safarians S., Barsky S. H. A novel strategy for the investigation of clonality in precancerous disease states and early stages of tumor progression. Biochem. Biophys. Res. Commun., 199: 511-518, 1994.[CrossRef][Medline]
  21. Dijkhuizen S. M., Entius M. M., Clement M. J., Polak M. M., Van den Berg F. M., Craanen M. E., Slebos R. J., Offerhaus G. J. Multiple hyperplastic polyps in the stomach: evidence for clonality and neoplastic potential. Gastroenterology, 112: 561-566, 1997.[CrossRef][Medline]
  22. Franklin W. A., Gazdar A. F., Haney J., Wistuba I. I., La Rosa F. G., Kennedy T., Ritchey D. M., Miller Y. E. Widely dispersed p53 mutation in respiratory epithelium. A novel mechanism for field carcinogenesis. J. Clin. Investig., 100: 2133-2137, 1997.[Medline]
  23. Wainscoat J. S., Fey M. F. Assessment of clonality in human tumors: a review. Cancer Res., 50: 1355-1360, 1990.[Abstract/Free Full Text]
  24. Nakashima H., Honda M., Inoue H., Shibuta K., Arinaga S., Era S., Ueo H., Mori M., Akiyoshi T. Microsatellite instability in multiple gastric cancers. Int. J. Cancer, 64: 239-242, 1995.[Medline]
  25. Garcia S. B., Novelli M., Wright N. A. The clonal origin and clonal evolution of epithelial tumours. Int. J. Exp. Pathol., 81: 89-116, 2000.[CrossRef][Medline]
  26. Dietrich C. U., Pandis N., Teixeira M. R., Bardi G., Gerdes A. M., Andersen J. A., Heim S. Chromosome abnormalities in benign hyperproliferative disorders of epithelial and stromal breast tissue. Int. J. Cancer, 60: 49-53, 1995.[Medline]
  27. Noguchi S., Motomura K., Inaji H., Imaoka S., Koyama H. Clonal analysis of solitary intraductal papilloma of the breast by means of polymerase chain reaction. Am. J. Pathol., 144: 1320-1325, 1994.[Abstract]
  28. Noguchi S., Motomura K., Inaji H., Imaoka S., Koyama H. Clonal analysis of predominantly intraductal carcinoma and precancerous lesions of the breast by means of polymerase chain reaction. Cancer Res., 54: 1849-1853, 1994.[Abstract/Free Full Text]
  29. Rosenberg C. L., Larson P. S., Romo J. D., De Las M. A., Faller D. V. Microsatellite alterations indicating monoclonality in atypical hyperplasias associated with breast cancer. Hum. Pathol., 28: 214-219, 1997.[CrossRef][Medline]
  30. Cardiff R. D., Fanning T. G., Morris D. W., Ashley R. L., Faulkin L. J. Restriction endonuclease studies of hyperplastic outgrowth lines from BALB/cfC3H mouse hyperplastic mammary nodules. Cancer Res., 41: 3024-3029, 1981.[Abstract/Free Full Text]
  31. Sandgren E. P., Schroeder J. A., Qui T. H., Palmiter R. D., Brinster R. L., Lee D. C. Inhibition of mammary gland involution is associated with transforming growth factor {alpha} but not c-myc-induced tumorigenesis in transgenic mice. Cancer Res., 55: 3915-3927, 1995.[Abstract/Free Full Text]
  32. Sandgren E. P., Luetteke N. C., Palmiter R. D., Brinster R. L., Lee D. C. Overexpression of TGF {alpha} in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell, 61: 1121-1135, 1990.[CrossRef][Medline]
  33. Jhappan C., Stahle C., Harkins R. N., Fausto N., Smith G. H., Merlino G. T. TGF {alpha} overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell, 61: 1137-1146, 1990.[CrossRef][Medline]
  34. Matsui Y., Halter S. A., Holt J. T., Hogan B. L., Coffey R. J. Development of mammary hyperplasia and neoplasia in MMTV-TGF {alpha} transgenic mice. Cell, 61: 1147-55, 1990.[CrossRef][Medline]
  35. Medina D. Preneoplastic lesions in mouse mammary tumorigenesis. Methods Cancer Res., 7: 3-53, 1973.
  36. Cardiff R. D. Protoneoplasia: the molecular biology of murine mammary hyperplasia. Adv. Cancer Res., 42: 167-190, 1984.[Medline]
  37. Kisseberth W. C., Brettingen N. T., Lohse J. K., Sandgren E. P. Ubiquitous expression of marker transgenes in mice and rats. Dev. Biol., 214: 128-138, 1999.[CrossRef][Medline]
  38. Neznanov N., Thorey I. S., Cecena G., Oshima R. G. Transcriptional insulation of the human keratin 18 gene in transgenic mice. Mol. Cell. Biol., 13: 2214-2223, 1993.[Abstract/Free Full Text]
  39. Thorey I. S., Meneses J. J., Neznanov N., Kulesh D. A., Pedersen R. A., Oshima R. G. Embryonic expression of human keratin 18 and K18-ß-galactosidase fusion genes in transgenic mice. Dev. Biol., 160: 519-534, 1993.[CrossRef][Medline]
  40. Clifton K. H., Kamiya K., Mulcahy R. T., Gould M. N. Radiogenic neoplasia in the thyroid and mammary clonogens: progress, problems and possibilities. Basic Life Sci., 33: 329-344, 1985.[Medline]
  41. Moser A. R., Mattes E. M., Dove W. F., Lindstrom M. J., Haag J. D., Gould M. N. ApcMin, a mutation in the murine Apc gene, predisposes to mammary carcinomas and focal alveolar hyperplasias. Proc. Natl. Acad. Sci. USA, 90: 8977-8981, 1993.[Abstract/Free Full Text]
  42. Braun K. M., Degen J. L., Sandgren E. P. Hepatocyte transplantation in a model of toxin-induced liver disease: variable therapeutic effect during replacement of damaged parenchyma by donor cells. Nat. Med., 6: 320-326, 2000.[CrossRef][Medline]
  43. Weglarz T. C., Degen J. L., Sandgren E. P. Hepatocyte transplantation into diseased mouse liver. Kinetics of parenchymal repopulation and identification of the proliferative capacity of tetraploid and octaploid hepatocytes. Am. J. Pathol., 157: 1963-1974, 2000.[Abstract/Free Full Text]
  44. Mintz B., Slemmer G. Gene control of neoplasia. I. Genotypic mosaicism in normal and preneoplastic mammary glands of allophenic mice. J. Natl. Cancer Inst. (Bethesda), 43: 87-109, 1969.
  45. Thomas G. A., Williams D., Williams E. D. The demonstration of tissue clonality by X-linked enzyme histochemistry. J. Pathol., 155: 101-108, 1988.[CrossRef][Medline]
  46. Tsai Y. C., Lu Y., Nichols P. W., Zlotnikov G., Jones P. A., Smith H. S. Contiguous patches of normal human mammary epithelium derived from a single stem cell: implications for breast carcinogenesis. Cancer Res., 56: 402-404, 1996.[Abstract/Free Full Text]
  47. Novelli M., Cossu A., Oukrif D., Quaglia A., Lakhani S., Poulsom R., Sasieni P., Carta P., Contini M., Pasca A., Palmieri G., Bodmer W., Tanda F., Wright N. X-inactivation patch size in human female tissue confounds the assessment of tumor clonality. Proc. Natl. Acad. Sci. USA, 100: 3311-3314, 2003.[Abstract/Free Full Text]
  48. Heim S., Teixeira M. R., Dietrich C. U., Pandis N. Cytogenetic polyclonality in tumors of the breast. Cancer Genet. Cytogenet., 95: 16-19, 1997.[CrossRef][Medline]
  49. Pandis N., Jin Y., Gorunova L., Petersson C., Bardi G., Idvall I., Johansson B., Ingvar C., Mandahl N., Mitelman F., Heim S. Chromosome analysis of 97 primary breast carcinomas: identification of eight karyotypic subgroups. Genes Chromosomes Cancer, 12: 173-185, 1995.[Medline]
  50. Novelli M. R., Williamson J. A., Tomlinson I. P., Elia G., Hodgson S. V., Talbot I. C., Bodmer W. F., Wright N. A. Polyclonal origin of colonic adenomas in an XO/XY patient with FAP. Science (Wash. DC), 272: 1187-1190, 1996.[Abstract]




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