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Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition

¹ Structural Cell Biology Unit, Institute of Medical Anatomy, The Panum Institute, DK-2200 Copenhagen N, Denmark
² Zoophysiological Laboratory, The August Krogh Institute, DK-2100 Copenhagen Ø, Denmark
³ Department of Pathology, Rigshospitalet, DK-2100 Copenhagen Ø, Denmark
⁴ Life Sciences Division, Berkeley National Laboratory, Berkeley, CA 94720, USA

View larger version (91K): [in a new window]   Fig. 1. Luminal epithelial cells form aberrant acini in collagen gels. Cells were embedded in either rBM (a,c) or hydrated collagen-I gels (b,d). (a,b) Phase-contrast microscopy revealed almost identical spherical structures in collagen-I and rBM albeit with no visible central lumen in spheres in collagen-I. (c,d) Haematoxylin staining of cryostat sections of the gels showed lumen formation only in rBM (bar, 25 µm).

View larger version (69K): [in a new window]   Fig. 2. (A) Luminal cells make inside-out acini in collagen. Luminal cells were double-stained either for (a) sialomucin (red) and ESA (green); (b) sialomucin (red) and occludin (green); (c) nuclear stain (red) and ß4-integrin (green); or (d) nuclear stain (red) and type IV collagen (green). Spheres in collagen-I gel (a',b') exhibit reversed polarity compared with cells in rBM (a,b), do not target ß4-integrin basolaterally (compare c and c') and fail to deposit a basement membrane (compare d and d'). (B) Reversal of inside-out acini by addition of myoepithelial cells. In the presence of myoepithelial cells (a''-d''), the polarity is corrected as is the endogenous BM deposition and integrin targeting. (bar, 25 µm).

View larger version (43K): [in a new window]   Fig. 3. Luminal epithelial cells and myoepithelial cells sort themselves out inside rBM gels but form bilayered acini inside collagen gels. The spatial organization of myoepithelial cells in relation to luminal epithelial acinar backbones was analyzed in rBM (a) and hydrated collagen-I gel (b) and compared to that in normal breast (c). Gels were doublestained for Thy-1 to demonstrate myoepithelial cells (green) and propidium iodide to demonstrate nuclei (red). Note that whereas myoepithelial cells segregate from luminal epithelial acini in rBM, in vivo-like double-layered structures are formed in the collagen-I based acinus assay (bar, 25 µm).

View larger version (16K): [in a new window]   Fig. 4. Only laminin-1 and not laminin-5 and -10/11 can correctly polarize acini in collagen gels. Luminal epithelial cells without myoepithelial (LEP alone) had no correctly polarized acini. Addition of 10% rBM or pure laminin-1 reversed polarity to the same degree as normal-derived myoepithelial cells (MEP). Neither affinity purified laminin-5 nor laminin-10/11 possessed the ability to revert acinus polarity.

View larger version (34K): [in a new window]   Fig. 5. LAMA-1 chain is expressed only in myoepithelial cells. RT-PCR analysis of laminin 1 (LAMA1), laminin 3 (LAMA3), laminin 5 (LAMA5), type IV collagen 1 (COL4A1) and type IV collagen 2 (COL4A2) in luminal epithelial cells in rBM and collagen-I gel and in myoepithelial cells and MCF-7 cells. GAPDH is used as an internal control. Note that the only laminin chain that is not expressed in the pure luminal epithelial cultures is the 1-chain.

View larger version (40K): [in a new window]   Fig. 6. Luminal epithelial cells and myoepithelial cells can deposit both 3-chain and 5-chain of laminin but only myoepithelial cells can deposit laminin-1 chain. Luminal epithelial cells (a,c,e) and myoepithelial cells (b,d,f) embedded in rBM were stained for laminin 1 (a,b; green), laminin 3 (c,d; green) and laminin 5 (e,f; green). Both luminal epithelial cells and myoepithelial cells deposit a basement membrane except for the lack of 1-chain deposition by the former. Nuclear stain, red. (bar, 25 µm).

View larger version (99K): [in a new window]   Fig. 7. Cancer-derived myoepithelial cells share many characteristics of normal myoepithelial cells but fail to reorient inside-out acini. (A) Isolation of cancer-derived myoepithelial cells expressing typical myoepithelial markers. Purified cancer-derived myoepithelial cells (a,b) stained with -smooth muscle actin (green) and keratins (red) to document the concurrent myo- and epithelial phenotypes (bar, 50 µm). (B) Lack of reversal of inside-out acini by cancer-derived myoepithelial cells. Sections of (a) the acinus assay embedded with the cancer-derived myoepithelial cell line and (b) an immortalized, normal-derived myoepithelial cell line. The sections are double stained for the apical marker sialomucin (green) and the nuclear stain propidium iodide (red). Note the lack of polarization with the cancer myoepithelial cells (C-MEP) and the correctly polarized lumina (encircled) with the immortalized myoepithelial cells (N-MEP) (bar, 25 µm).

View larger version (53K): [in a new window]   Fig. 8. The cancer-derived myoepithelial cell line lacks the expression of laminin-1 chain only. RT-PCR on RNA extracted from cancer-derived myoepithelial cell line, immortalized, normal-derived myoepithelial cells (MEP), primary cultured MEP and MCF-7 S9 cells. The panel shows 1-chain (LAMA1), 3-chain (LAMA3), 5-chain (LAMA5) and GAPDH as an internal control. While the 3 mRNA was low in the cancer-derived MEP cell line, the l-chain was the only differentially expressed chain between normal-derived and cancer-derived myoepithelial cells.

View larger version (20K): [in a new window]   Fig. 9. Only one of four cancer-derived myoepithelial cells can completely reverse acinus polarity in collagen gels. Frequency of correctly polarized acini in sections of collagen-I gels. Addition of cancer-derived myoepithelial cells from four different sources failed completely to revert acini in two instances, had a partial impact on reversion in one instance, and could revert in the fourth case. Inserts show staining for ß4-integrin (green) counterstained with propidium iodide (red). Data represent mean (±s.e.m.) except for the primary cancer-derived MEP, which are means of triplicate determinations (±s.d.). (bar, 20 µm).

View larger version (156K): [in a new window]   Fig. 10. Reduced staining of laminin-1 in human breast cancer. Cryostat sections of terminal duct lobular unit (TDLU) (a), a ductal carcinoma in situ (DCIS) (b) and four different infiltrating ductal breast carcinomas (IDC) (c-f) stained with immunoperoxidase for laminin-1 and counterstained with hematoxylin. In the TDLU a strongly stained, continuous basement membrane is delineated at the stromal (S)-epithelial (E) interface (a). In carcinomas, laminin-1 staining is localized to the stromal (S)-cancer epithelial (C) interface, but is discontinuous and less pronounced. (bar, 25 µm).

View larger version (106K): [in a new window]   Fig. 11. Absence of, and reduced staining of laminin-1 in tumor-associated myoepithelial cells. Double-labeling immunofluorescence of a normal acinus in a TDLU (a,b) and two different carcinomas (c,d and e,f) to demonstrate laminin-1 in red (a,c,e) and in a coexposure with myoepithelial cells in green (b,d,f) as stained with keratin 17 (b,d) or maspin (f). The bottom panels illustrate the presence of myoepithelial cells at the stromal (S)-cancer epithelial (C) interface in total absence of laminin-1 staining. The middle panels show infiltrating ductal carcinoma with tumor-associated myoepithelial cells expressing a variable but generally weak staining for laminin-1 compared with a normal acinus in the top row. (bar, 50 µm).