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. 2010 Feb;176(2):827-38.
doi: 10.2353/ajpath.2010.090006. Epub 2009 Dec 30.

Quantitative analysis of three-dimensional human mammary epithelial tissue architecture reveals a role for tenascin-C in regulating c-met function

Agne Taraseviciute  1 , Benjamin T Vincent, Pepper Schedin, Peter Lloyd Jones
Affiliations

Quantitative analysis of three-dimensional human mammary epithelial tissue architecture reveals a role for tenascin-C in regulating c-met function

Agne Taraseviciute et al. Am J Pathol. 2010 Feb.

Abstract

Remodeling of the stromal extracellular matrix and elevated expression of specific proto-oncogenes within the adjacent epithelium represent cardinal features of breast cancer, yet how these events become integrated is not fully understood. To address this question, we focused on tenascin-C (TN-C), a stromal extracellular matrix glycoprotein whose expression increases with disease severity. Initially, nonmalignant human mammary epithelial cells (MCF-10A) were cultured within a reconstituted basement membrane (BM) where they formed three-dimensional (3-D) polarized, growth-attenuated, multicellular acini, enveloped by a continuous endogenous BM. In the presence of TN-C, however, acini failed to generate a normal BM, and net epithelial cell proliferation increased. To quantify how TN-C alters 3-D tissue architecture and function, we developed a computational image analysis algorithm, which showed that although TN-C disrupted acinar surface structure, it had no effect on their volume. Thus, TN-C promoted epithelial cell proliferation leading to luminal filling, a process that we hypothesized involved c-met, a proto-oncogene amplified in breast tumors that promotes intraluminal filling. Indeed, TN-C increased epithelial c-met expression and promoted luminal filling, whereas blockade of c-met function reversed this phenotype, resulting in normal BM deposition, proper lumen formation, and decreased cell proliferation. Collectively, these studies, combining a novel quantitative image analysis tool with 3-D organotypic cultures, demonstrate that stromal changes associated with breast cancer can control proto-oncogene function.

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Figures

Figure 1
Figure 1
Stromal TN-C alters normal 3-D mammary epithelial tissue architecture. A: Tissue sections from normal human mammary gland (top), DCIS (middle), and IDC (lower) stained with hematoxylin and eosin (H&E) (left) or TN-C (right). Scale bar, 50 μm. B: Morphology of MCF-10A acini generated in Matrigel in the absence (top) or presence (bottom) of TN-C for 8 days (phase contrast, left). Confocal immunofluorescence staining with laminin-V (green; middle and right), Ki-67 (red; middle), and cleaved caspase-3 (red; right) in 8 day cultures. Asterisk indicates loss of a continuous BM (middle) and position of cells residing outside of the BM zone (right) in the presence of TN-C. Scale bars, 50 μm. C: Quantification of Ki-67 immunoreactivity in MCF-10A acini revealed a 1.6-fold increase in proliferation (n = 66 acini, *P < 0.009), yet no differences in apoptosis (quantification of cleaved caspase-3 immunoreactivity; n = 40 acini, P = 0.68) in response to TN-C.
Figure 2
Figure 2
Reconstructing 3-D mammary epithelial tissue architecture. A: Schema delineating the computational steps used to measure acinar surface roughness and volume. B: In this example, the equatorial 2-D confocal slice from a laminin-V immunostained acinus, treated previously with TN-C for 8 days, was subjected to dx and dy pre-processing based on fluorescence intensity (top). Active contours were manually initiated by selecting multiple points close to the edge of the acinus (lower left). After active contour fitting, a final trace of the edge was obtained automatically (lower right). C: The trace obtained in B was extrapolated to all subsequent remaining 2-D slices of the acinus resulting in the automated generation of a montage of traces. D: Montages described in C were used to render a 3-D projection.
Figure 3
Figure 3
TN-C increases surface roughness, but not acinar volume. A: Examples of 3-D renditions of individual acini (black) generated in the absence (top) or presence (bottom) of TN-C in 8 day cultures. A customized ellipsoid (red-yellow) was designed and fitted to each individual acinus. B: Change in radius for each acinar slice (black) denotes distance away from the perfect ellipsoid for the acini depicted in A; the red horizontal line at ‘0′ represents the perfect ellipsoid. In the absence of TN-C (top), each slice did not deviate more than a few μms from the perfect ellipsoid, whereas in the presence of TN-C (bottom), deviations exceeded 10 μm. C: 3-D acinar structure was quantified by measuring root mean square (RMS; absolute difference from perfect ellipsoid) values for acini cultured for 8 days in the absence or presence of TN-C. TN-C evoked a 1.7-fold increase in RMS (−TN-C = 2.98 ± 0.18, +TN-C = 4.96 ± 0.38, n = 103; P = 0.024). D: Mercator projections of 3-D acini: protrusions are displayed in yellow and red while indentations appear blue. E: Distribution of RMS values (left) and volumes (right) for acini generated in the absence (dashed line) or presence (solid line) of TN-C for 8 days. (F) Plotting RMS versus volume yields 2 distinct groups of acini with the line showing the best linear classification using logistic regression at a performance of 79.5%.
Figure 4
Figure 4
TN-C promotes luminal filling and upregulates c-met. A: Confocal immunofluorescence staining with laminin-V (green; upper) and DAPI (nuclei, blue) revealed changes in lumen structure (white dotted lines) in acini cultured with or without TN-C. C-met staining intensity is increased in the presence of TN-C (lower right) when compared with control (lower left). Scale bars, upper panels, 50 μm; lower panels, 25 μm. B: Western blot analysis confirmed that c-met levels are increased in the presence of TN-C, whereas secreted HGF levels remain unchanged. GAPDH is the loading control. C: Densitometric analyses of c-met levels relative to GAPDH reveal a significant 1.8-fold increase in the presence of TN-C. D: Tissue sections from the normal human mammary gland (top) and grade 2 IDCs (middle and bottom) stained with H&E (left) or c-met and TN-C (red and blue, respectively; right). Scale bar, 50 μm.
Figure 5
Figure 5
Blocking c-met function reverses the TN-C–dependent phenotype. A: Representative confocal immunofluorescence photomicrographs showing reversion of the TN-C–induced phenotype on introduction of a function-blocking c-met antibody. B: C-met blockade significantly decreases the proliferation of MCF-10A acini treated with TN-C (−TN-C + IgG, 7.82 ± 1.49; +TN-C + IgG, 13.65 ± 1.82; +TN-C + α-c-met, 7.12 ± 1.41; n = 60; *P = 0.017; **P = 0.0079). C: Examples of 3-D renditions of individual acini (black), generated in the absence of TN-C (left), presence of TN-C (middle), and presence of TN-C and c-met blocking antibody (right), along with customized ellipsoids (red-yellow). D: C-met blockade restores normal acinar architecture as measured by RMS (−TN-C + IgG, 2.95 ± 0.28; +TN-C + IgG, 3.89 ± 0.39; +TN-C + α-c-met, 2.97 ± 0.29; n = 74; *P = 0.036). E: Distribution of RMS values (left) and volumes (right) for acini generated in the absence of TN-C (solid line), presence of TN-C (horizontally dashed line), and in the presence of TN-C and c-met blocking antibody (vertically dashed line) for 8 days. F: Plotting volume versus RMS results in clustering of the 3 groups of acini into 2 distinct populations, (ie, the absence of TN-C and presence of TN-C and c-met blocking antibody form one cluster, whereas the presence of TN-C and IgG form a different cluster), with the line showing the best linear classification using logistic regression at a performance of 72%.
Figure 6
Figure 6
Hypothetical schema delineating how stromal TN-C promotes epithelial cell proliferation and intraluminal filling within the mammary epithelium via up-regulation of c-met.
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References

    1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70. - PubMed
    1. Bissell MJ, Radisky D. Putting tumours in context. Nat Rev Cancer. 2001;1:46–54. - PMC - PubMed
    1. Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, Gefen A, Reinhart-King CA, Margulies SS, Dembo M, Boettiger D, Hammer DA, Weaver VM. Tensional homeostasis and the malignant phenotype. Cancer Cell. 2005;8:241–254. - PubMed
    1. Schedin P, Mitrenga T, McDaniel S, Kaeck M. Mammary ECM composition and function are altered by reproductive state. Mol Carcinog. 2004;41:207–220. - PubMed
    1. Aggeler J, Ward J, Blackie LM, Barcellos-Hoff MH, Streuli CH, Bissell MJ. Cytodifferentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to development in vivo, J Cell Sci. 1991;99 (Pt 2):407–417. - PubMed

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