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Mammary carcinoma behavior is programmed in the precancer stem cell.

Damonte P, Hodgson JG, Chen JQ, Young LJ, Cardiff RD, Borowsky AD - Breast Cancer Res. (2008)

Bottom Line: No changes are associated with progression, although spontaneous focal amplifications and deletions were detected occasionally.Telomerase activity is increased in both the MINO and the derived tumors when compared with normal mouse mammary gland.Within the context of this model, these cells have programmed potential for latency and metastasis that does not appear to require sequential genetic 'hits' for transformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Center for Comparative Medicine, UC Davis, County Road 98 and Hutchison Drive, Davis, California 95616, USA.

ABSTRACT

Introduction: The 'MINO' (mammary intraepithelial neoplasia outgrowth) mouse model of ductal carcinoma in situ (DCIS) consists of six lines with distinct morphologic phenotypes and behavior, each meeting experimentally defined criteria for 'precancer'. Specifically, these lines grow orthotopically in cleared mammary fat pads and consistently progress to an invasive phenotype that is capable of ectopic growth. Transition to carcinoma has a consistent latency for each line, and three of the lines also exhibit pulmonary metastatic potential.

Methods: Gland cleared orthotopic transplanted precancer MINO tissues were analyzed by bacterial artifical chromosome and oligo array comparative genomic hybridization, microsatellite PCR, and telomerase repeat amplification assay. MINO cells were dissociated and cultured in three dimensional culture and transplanted in syngeneic gland cleared mammary fat pads.

Results: Comparative genomic hybridization shows that the precancer and invasive tumors are genetically stable, with low level changes including whole chromosome gains in some lines. No changes are associated with progression, although spontaneous focal amplifications and deletions were detected occasionally. Microsatellite analysis shows a low frequency of alterations that are predominantly permanent within a MINO line. Telomerase activity is increased in both the MINO and the derived tumors when compared with normal mouse mammary gland. Dissociation of the precancer lesion cells and three dimensional 'spheroid' culture of single cells reveals a bipotential for myoepithelial and luminal differentiation and the formation of unique three-dimensional 'MINOspheres'. These MINOspheres exhibit features that are intermediate between spheroids that are derived from normal and carcinoma cells. Transplantation of a single cell derived MINOsphere recapitulates the outgrowth of the precancer morphology and progression to carcinoma.

Conclusion: These data establish a precancer 'stem' cell that is capable of self-renewal and multilineage differentiation as the origin of invasive cancer. Within the context of this model, these cells have programmed potential for latency and metastasis that does not appear to require sequential genetic 'hits' for transformation.

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Three-dimensional culture of single cells from normal prelactating mammary gland, MINO precancer, and invasive carcinoma. (a, d, g) Inverted microscope phase contrast images as well as histologic images generated from paraffin-embedded 4 μm sections of the three-dimensional cultures stained by immunohistochemistry with (b, e, h) CK8–18 or (c, f, i) CK14 are shown. The magnification scale (lower right panel i) is identical for all histology panels and approximate for the inverted microscopy photographs. CK8–18 confirms that the major cell population is a luminal phenotype, but CK14 shows that there is also myoepithelial differentiation of single cells, documenting bipotential of the individual cells giving rise to these three-dimensional structures. A single MINOsphere from line 4w4 was transplanted from the three dimensional culture into the gland cleared fat pad of a 3-week-old female FVB/n mouse. The mammary gland was removed 10 weeks after transplant and a whole mount mammary gland preparation was made with hematoxylin stain to visualize cell density (j). After photography, the same gland was processed for histologic sectioning, and the resulting 4 μm hematoxylin and eosin stained tissue section is shown (k). At least three foci of tumor are seen in the differentiation zone of the MINO. One is indicated by the arrow. CK, cytokeratin; MINO, mammary intraepithelial neoplasia outgrowth.
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Figure 3: Three-dimensional culture of single cells from normal prelactating mammary gland, MINO precancer, and invasive carcinoma. (a, d, g) Inverted microscope phase contrast images as well as histologic images generated from paraffin-embedded 4 μm sections of the three-dimensional cultures stained by immunohistochemistry with (b, e, h) CK8–18 or (c, f, i) CK14 are shown. The magnification scale (lower right panel i) is identical for all histology panels and approximate for the inverted microscopy photographs. CK8–18 confirms that the major cell population is a luminal phenotype, but CK14 shows that there is also myoepithelial differentiation of single cells, documenting bipotential of the individual cells giving rise to these three-dimensional structures. A single MINOsphere from line 4w4 was transplanted from the three dimensional culture into the gland cleared fat pad of a 3-week-old female FVB/n mouse. The mammary gland was removed 10 weeks after transplant and a whole mount mammary gland preparation was made with hematoxylin stain to visualize cell density (j). After photography, the same gland was processed for histologic sectioning, and the resulting 4 μm hematoxylin and eosin stained tissue section is shown (k). At least three foci of tumor are seen in the differentiation zone of the MINO. One is indicated by the arrow. CK, cytokeratin; MINO, mammary intraepithelial neoplasia outgrowth.

Mentions: A minority of single cells in each group gave rise to three-dimensional growths in culture. In the normal mammary gland controls, about 1% to 9% of cells gave rise to small spheroids that grew in size gradually but retained a spherical shape (spheroids), with a hollow center appearing after the sphere reached a sufficient size for identification (Figure 3a). The morphology of the spheroids and the percentage of cells that are capable of this growth were unchanged between virgin (multiple glands used from multiple mice) and prelactating normal mammary gland cells. Histologic sections of these normal spheroids revealed a clear double layer of cells with uniform, larger luminal mammary epithelial cells lining the center of a hollow cavity, surrounded by a second layer of flatter cells with smaller nuclei forming a discontinuous web.


Mammary carcinoma behavior is programmed in the precancer stem cell.

Damonte P, Hodgson JG, Chen JQ, Young LJ, Cardiff RD, Borowsky AD - Breast Cancer Res. (2008)

Three-dimensional culture of single cells from normal prelactating mammary gland, MINO precancer, and invasive carcinoma. (a, d, g) Inverted microscope phase contrast images as well as histologic images generated from paraffin-embedded 4 μm sections of the three-dimensional cultures stained by immunohistochemistry with (b, e, h) CK8–18 or (c, f, i) CK14 are shown. The magnification scale (lower right panel i) is identical for all histology panels and approximate for the inverted microscopy photographs. CK8–18 confirms that the major cell population is a luminal phenotype, but CK14 shows that there is also myoepithelial differentiation of single cells, documenting bipotential of the individual cells giving rise to these three-dimensional structures. A single MINOsphere from line 4w4 was transplanted from the three dimensional culture into the gland cleared fat pad of a 3-week-old female FVB/n mouse. The mammary gland was removed 10 weeks after transplant and a whole mount mammary gland preparation was made with hematoxylin stain to visualize cell density (j). After photography, the same gland was processed for histologic sectioning, and the resulting 4 μm hematoxylin and eosin stained tissue section is shown (k). At least three foci of tumor are seen in the differentiation zone of the MINO. One is indicated by the arrow. CK, cytokeratin; MINO, mammary intraepithelial neoplasia outgrowth.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2481504&req=5

Figure 3: Three-dimensional culture of single cells from normal prelactating mammary gland, MINO precancer, and invasive carcinoma. (a, d, g) Inverted microscope phase contrast images as well as histologic images generated from paraffin-embedded 4 μm sections of the three-dimensional cultures stained by immunohistochemistry with (b, e, h) CK8–18 or (c, f, i) CK14 are shown. The magnification scale (lower right panel i) is identical for all histology panels and approximate for the inverted microscopy photographs. CK8–18 confirms that the major cell population is a luminal phenotype, but CK14 shows that there is also myoepithelial differentiation of single cells, documenting bipotential of the individual cells giving rise to these three-dimensional structures. A single MINOsphere from line 4w4 was transplanted from the three dimensional culture into the gland cleared fat pad of a 3-week-old female FVB/n mouse. The mammary gland was removed 10 weeks after transplant and a whole mount mammary gland preparation was made with hematoxylin stain to visualize cell density (j). After photography, the same gland was processed for histologic sectioning, and the resulting 4 μm hematoxylin and eosin stained tissue section is shown (k). At least three foci of tumor are seen in the differentiation zone of the MINO. One is indicated by the arrow. CK, cytokeratin; MINO, mammary intraepithelial neoplasia outgrowth.
Mentions: A minority of single cells in each group gave rise to three-dimensional growths in culture. In the normal mammary gland controls, about 1% to 9% of cells gave rise to small spheroids that grew in size gradually but retained a spherical shape (spheroids), with a hollow center appearing after the sphere reached a sufficient size for identification (Figure 3a). The morphology of the spheroids and the percentage of cells that are capable of this growth were unchanged between virgin (multiple glands used from multiple mice) and prelactating normal mammary gland cells. Histologic sections of these normal spheroids revealed a clear double layer of cells with uniform, larger luminal mammary epithelial cells lining the center of a hollow cavity, surrounded by a second layer of flatter cells with smaller nuclei forming a discontinuous web.

Bottom Line: No changes are associated with progression, although spontaneous focal amplifications and deletions were detected occasionally.Telomerase activity is increased in both the MINO and the derived tumors when compared with normal mouse mammary gland.Within the context of this model, these cells have programmed potential for latency and metastasis that does not appear to require sequential genetic 'hits' for transformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Center for Comparative Medicine, UC Davis, County Road 98 and Hutchison Drive, Davis, California 95616, USA.

ABSTRACT

Introduction: The 'MINO' (mammary intraepithelial neoplasia outgrowth) mouse model of ductal carcinoma in situ (DCIS) consists of six lines with distinct morphologic phenotypes and behavior, each meeting experimentally defined criteria for 'precancer'. Specifically, these lines grow orthotopically in cleared mammary fat pads and consistently progress to an invasive phenotype that is capable of ectopic growth. Transition to carcinoma has a consistent latency for each line, and three of the lines also exhibit pulmonary metastatic potential.

Methods: Gland cleared orthotopic transplanted precancer MINO tissues were analyzed by bacterial artifical chromosome and oligo array comparative genomic hybridization, microsatellite PCR, and telomerase repeat amplification assay. MINO cells were dissociated and cultured in three dimensional culture and transplanted in syngeneic gland cleared mammary fat pads.

Results: Comparative genomic hybridization shows that the precancer and invasive tumors are genetically stable, with low level changes including whole chromosome gains in some lines. No changes are associated with progression, although spontaneous focal amplifications and deletions were detected occasionally. Microsatellite analysis shows a low frequency of alterations that are predominantly permanent within a MINO line. Telomerase activity is increased in both the MINO and the derived tumors when compared with normal mouse mammary gland. Dissociation of the precancer lesion cells and three dimensional 'spheroid' culture of single cells reveals a bipotential for myoepithelial and luminal differentiation and the formation of unique three-dimensional 'MINOspheres'. These MINOspheres exhibit features that are intermediate between spheroids that are derived from normal and carcinoma cells. Transplantation of a single cell derived MINOsphere recapitulates the outgrowth of the precancer morphology and progression to carcinoma.

Conclusion: These data establish a precancer 'stem' cell that is capable of self-renewal and multilineage differentiation as the origin of invasive cancer. Within the context of this model, these cells have programmed potential for latency and metastasis that does not appear to require sequential genetic 'hits' for transformation.

Show MeSH
Related in: MedlinePlus