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Effects of Noninhibitory Serpin Maspin on the Actin Cytoskeleton: A Quantitative Image Modeling Approach

  • Mohammed Al-Mamun (a1) (a2), Lorna Ravenhill (a3), Worawut Srisukkham (a1), Alamgir Hossain (a1) (a4), Charles Fall (a1), Vincent Ellis (a3) and Rosemary Bass (a5)...


Recent developments in quantitative image analysis allow us to interrogate confocal microscopy images to answer biological questions. Clumped and layered cell nuclei and cytoplasm in confocal images challenges the ability to identify subcellular compartments. To date, there is no perfect image analysis method to identify cytoskeletal changes in confocal images. Here, we present a multidisciplinary study where an image analysis model was developed to allow quantitative measurements of changes in the cytoskeleton of cells with different maspin exposure. Maspin, a noninhibitory serpin influences cell migration, adhesion, invasion, proliferation, and apoptosis in ways that are consistent with its identification as a tumor metastasis suppressor. Using different cell types, we tested the hypothesis that reduction in cell migration by maspin would be reflected in the architecture of the actin cytoskeleton. A hybrid marker-controlled watershed segmentation technique was used to segment the nuclei, cytoplasm, and ruffling regions before measuring cytoskeletal changes. This was informed by immunohistochemical staining of cells transfected stably or transiently with maspin proteins, or with added bioactive peptides or protein. Image analysis results showed that the effects of maspin were mirrored by effects on cell architecture, in a way that could be described quantitatively.


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Al-Mamun, M.A., Brown, L.J., Hossain, M.A., Fall, C., Wagstaff, L. & Bass, R. (2013). A hybrid computational model for the effects of maspin on cancer cell dynamics. J Theor Biol 337, 150160.
Bass, R., Moreno Fernandez, A.M. & Ellis, V. (2002). Maspin inhibits cell migration in the absence of protease inhibitory activity. J Biol Chem 277(49), 4684546848.
Bass, R., Wagstaff, L., Ravenhill, L. & Ellis, V. (2009). Binding of extracellular maspin to beta1 integrins inhibits vascular smooth muscle cell migration. J Biol Chem 284(40), 2771227720.
Bernardo, V., Lourenço, S.Q., Cruz, R., Monteiro-Leal, L.H., Silva, L.E., Camisasca, D.R., Farina, M. & Lins, U. (2009). Reproducibility of immunostaining quantification and description of a new digital image processing procedure for quantitative evaluation of immunohistochemistry in pathology. Microsc Microanal 15(4), 353365.
Bodenstine, T.M., Seftor, R.E., Khalkhali-Ellis, Z., Seftor, E.A., Pemberton, P.A. & Hendrix, M.J. (2012). Maspin: Molecular mechanisms and therapeutic implications. Cancer Metastasis Rev 31(3–4), 529551.
Breu, H., Gil, J., Kirkpatrick, D. & Werman, M. (1995). Linear time Euclidean distance transform algorithms. IEEE Trans Pattern Anal Mach Intell 17, 529533.
Cates, J.E., Whitaker, R.T. & Jones, G.M. (2005). Case study: an evaluation of user-assisted hierarchical watershed segmentation. Med Image Anal 9(6), 566578.
Cella, N., Contreras, A., Latha, K., Rosen, J.M. & Zhang, M. (2006). Maspin is physically associated with (beta)1 integrin regulating cell adhesion in mammary epithelial cells. FASEB J 20(9), 15101512.
Chen, E.I., Florens, L., Axelrod, F.T., Monosov, E., Barbas, C.F. III, Yates, J.R. III, Felding-Habermann, B. & Smith, J.W. (2005). Maspin alters the carcinoma proteome. FASEB J 19, 11231124.
Cheng, J. & Rajapakse, J.C. (2009). Segmentation of clustered nuclei with shape markers and marking function. IEEE Trans Biomed Eng 56(3), 741748.
Deshpande, M., Notari, L., Subramanian, P., Notario, V. & Becerra, S.P. (2012). Inhibition of tumor cell surface ATP synthesis by pigment epithelium-derived factor: implications for antitumor activity. Int J Oncol 41(1), 219227.
Fatima, M.M. & Seenivasagam, V. (2011). A fast fuzzy-c means based marker controlled watershed segmentation of clustered nuclei. In Proceedings of International Conference on Computer, Communication and Electrical Technology, 15–17 September 2011, Adriatic Islands, Split, Croatia.
Fouard, C. & Gedda, M. (2006). An objective comparison between grey weighted distance transforms and weighted distance transforms on curved spaces. In Discrete Geometry for Computer Imagery, Hutchison, D., Kanade, T., Kittler, J., et al. (Eds.), pp. 259–270. Lecture Notes in Computer Science, Springer.
Fuseler, J.W., Bedenbaugh, A., Yekkala, K. & Baudino, T.A. (2010). Fractal and image analysis of the microvasculature in normal intestinal submucosa and intestinal polyps in Apc(Min/+) mice. Microsc Microanal 16(1), 7379.
Fuseler, J.W., Millette, C.F., Davis, J.M. & Carver, W. (2007). Fractal and image analysis of morphological changes in the actin cytoskeleton of neonatal cardiac fibroblasts in response to mechanical stretch. Microsc Microanal 13, 133143.
Ghosh, M., Das, D., Chakraborty, C. & Ray, A.K. (2010). Automated leukocyte recognition using fuzzy divergence. Micron 41, 840846.
Gonzalez, R.C. & Woods, R.E. (1992). Digital imaging processing, Massachusetts: Addison-Wesley.
Jordan, P.A. & Gibbins, J.M. (2006). Extracellular disulfide exchange and the regulation of cellular function. Antioxid Redox Signal 8(3–4), 312324.
Jung, C. & Kim, C. (2010). Segmenting clustered nuclei using h-minima transform-based marker extraction and contour parameterization. IEEE Trans Biomed Eng 57(10), 26002604.
Ko, B.C., Gim, J. & Nam, J. (2011). Automatic white blood cell segmentation using stepwise merging rules and gradient vector flow snake. Micron 42, 695705.
Koyuncu, C.F., Arslan, S., Durmaz, I., Cetin-Atalay, R. & Gunduz-Demir, C. (2012). Smart markers for watershed-based cell segmentation. PLoS One 7(11), e48664.
Lara, H., Wang, Y., Beltran, A.S., Juárez-Moreno, K., Yuan, X., Kato, S., Leisewitz, A.V., Cuello Fredes, M., Licea, A.F., Connolly, D.C., Huang, L. & Blancafort, P. (2012). Targeting serous epithelial ovarian cancer with designer zinc finger transcription factors. J Biol Chem 287(35), 2987329886.
Liao, X.H., Li, Y.Q., Wang, N., Zheng, L., Xing, W.J., Zhao, D.W., Yan, T.B., Wang, Y., Liu, L.Y., Sun, X.G., Hu, P., Zhou, H. & Zhang, T.C. (2014). Re-expression and epigenetic modification of maspin induced apoptosis in MCF-7 cells mediated by myocardin. Cell Signal 26(6), 13351346.
Lindblad, J., Wählby, C., Bengtsson, E. & Zaltsman, A. (2004). Image analysis for automatic segmentation of cytoplasms and classification of Rac1 activation. Cytometry A 57(1), 2233.
Meddens, M.B., Rieger, B., Figdor, C.G., Cambi, A. & van den Dries, K. (2013). Automated podosome identification and characterization in fluorescence microscopy images. Microsc Microanal 19(1), 180189.
Mohapatra, S., Patra, D. & Satpathy, S. (2011). Automated leukemia detection in blood microscopic images using statistical texture analysis. In Proceedings of International Conference on Communication, Computing; Security, 12–14 February 2011, National Institute of Technology Rourkela, Odisha, India.
Mueller, J.L., Harmany, Z.T., Mito, J.K., Kennedy, S.A., Kim, Y., Dodd, L., Geradts, J., Kirsch, D.G., Willett, R.M., Brown, J.Q. & Ramanujam, N. (2013). Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Application to the detection of residual disease in tumor margins. PLoS One 8(6), e66198.
Ng, H.P., Ong, S.H., Foong, K.W., Goh, P.S. & Nowinski, W.L. (2008). Masseter segmentation using an improved watershed algorithm with unsupervised classification. Comput Biol Med 38(2), 171184.
Notari, L., Arakaki, N., Mueller, D., Meier, S., Amaral, J. & Becerra, S.P. (2010). Pigment epithelium-derived factor binds to cell-surface F(1)-ATP synthase. FEBS J 277(9), 21922205.
Nyirenda, N., Farkas, D.L. & Ramanujan, V.K. (2011). Preclinical evaluation of nuclear morphometry and tissue topology for breast carcinoma detection and margin assessment. Breast Cancer Res Treat 126(2), 345354.
Odero-Marah, V.A., Khalkhali-Ellis, Z., Chunthapong, J., Amir, S., Seftor, R.E., Seftor, E.A. & Hendrix, M.J. (2003). Maspin regulates different signaling pathways for motility and adhesion in aggressive breast cancer cells. Cancer Biol Ther 2, 398403.
Pham, T.D. (2008). Fuzzy fractal analysis of molecular imaging data. Proc IEEE 96(8), 13321347.
Plissiti, M.E., Nikou, C. & Charchanti, A. (2011). Combining shape, texture and intensity features for cell nuclei extraction in pap smear images. Pattern Recognit Lett 32, 838853.
Qin, L. & Zhang, M. (2010). Maspin regulates endothelial cell adhesion and migration through an integrin signaling pathway. J Biol Chem 285(42), 3236032369.
Ravenhill, L., Wagstaff, L., Edwards, D.R., Ellis, V. & Bass, R. (2010). The G-helix of maspin mediates effects on cell migration and adhesion. J Biol Chem 285(47), 3628536292.
Ridley, A.J., Schwartz, M.A., Burridge, K., Firtel, R.A., Ginsberg, M.H., Borisy, G., Parsons, J.T. & Horwitz, A.R. (2003). Cell migration: Integrating signals from front to back. Science 302(5651), 17041709.
Rosenfeld, A. & Pfaltz, J.L. (1968). Distance functions on digital pictures. Pattern Recognit 1(1), 3361.
Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7), 671675.
Seftor, R.E., Seftor, E.A., Sheng, S., Pemberton, P.A., Sager, R. & Hendrix, M.J. (1998). Maspin suppresses the invasive phenotype of human breast carcinoma. Cancer Res 58(24), 56815685.
Sheng, S., Carey, J., Seftor, E.A., Dias, L., Hendrix, M.J. & Sager, R. (1996). Maspin acts at the cell membrane to inhibit invasion and motility of mammary and prostatic cancer cells. Proc Natl Acad Sci USA 93(21), 1166911674.
Shi, H.Y., Stafford, L.J., Liu, Z., Liu, M. & Zhang, M. (2007). Maspin controls mammary tumor cell migration through inhibiting Rac1 and Cdc42, but not the RhoA GTPase. Cell Motil Cytoskeleton 64(5), 338346.
Shi, H.Y., Zhang, W., Liang, R., Abraham, S., Kittrell, F.S., Medina, D. & Zhang, M. (2001). Blocking tumor growth, invasion, and metastasis by maspin in a syngeneic breast cancer model. Cancer Res 61, 69456951.
Sinha, N. & Ramakrishnan, A.G. (2003). Automation of differential blood count. In Proceedings of TENCON Conference on Convergent Technologies for Asia-Pacific Region, 14–17 October 2003, Bangalore, India.
Srisukkham, W., Lepcha, P., Hossain, M.A., Zhang, L., Jiang, R. & Lim, H.N. (2013). A mobile enabled intelligent scheme to identify blood cancer for remote areas—Cell membrane segmentation using marker controlled watershed segmentation phase. In The 7th International Conference on Software, Knowledge, Information Management and Applications (SKIMA), Chiang Mai, Thailand, December 18–20.
Sun, H.Q. & Luo, Y.J. (2009). Adaptive watershed segmentation of binary particle image. J Microsc 233(2), 326330.
Teera-Umpon, N. (2005). Patch-based white blood cell nucleus segmentation using fuzzy clustering. ECTI Trans Electr Electron Commun 3, 510.
Teoh, S.S., Vieusseux, J., Prakash, M., Berkowicz, S., Luu, J., Bird, C.H., Law, R.H., Rosado, C., Price, J.T., Whisstock, J.C. & Bird, P.I. (2014). Maspin is not required for embryonic development or tumor suppression. Nat Commun 5, 3164.
Wählby, C., Sintorn, I.M., Erlandsson, F., Borgefors, G. & Bengtsson, E. (2004). Combining intensity, edge and shape information for 2D and 3D segmentation of cell nuclei in tissue sections. J Microsc 215(Pt 1), 6776.
Wienert, S., Heim, D., Saeger, K., Stenzinger, A., Beil, M., Hufnagl, P., Dietel, M., Denkert, C. & Klauschen, F. (2011). Detection and segmentation of cell nuclei in virtual microscopy images: A minimum-model approach. Sci Rep 2, 503.
Yang, L., Tuzel, O., Meer, P. & Foran, D. (2008). Automatic image analysis of histopathology specimens using concave vertex graph. In Proceedings of Medical Image Computing and Computer-Assisted Intervention, New York, NY, pp. 833–841.
Yin, S., Li, X., Meng, Y., Finley, R.L. Jr, Sakr, W., Yang, H., Reddy, N. & Sheng, S. (2005). Tumor-suppressive maspin regulates cell response to oxidative stress by direct interaction with glutathione S-transferase. J Biol Chem 280(41), 3498534996.
Zaritsky, A., Natan, S., Horev, J., Hecht, I., Wolf, L., Ben-Jacob, E. & Tsarfaty, I. (2011). Cell motility dynamics: A novel segmentation algorithm to quantify multi-cellular bright field microscopy images. PLoS One 6(11), e27593.
Zimmer, C. & Olivo-Marin, J.C. (2005). Coupled parametric active contours. IEEE Trans Pattern Anal Mach Intell 27(11), 18381842.
Zou, Z., Anisowicz, A., Hendrix, M.J., Thor, A., Neveu, M., Sheng, S., Rafidi, K., Seftor, E. & Sager, R. (1994). Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science 263(5146), 526529.
Zuiderveld, K. (1994). Contrast limited adaptive histogram equalization. In Graphics Gems IV , Heckbert, P.S. (Ed.), pp. 474485. Pittsburgh: Academic Press Professional, Inc.


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