Multiple cell types form specialized protein complexes that are used by the cell to actively degrade the surrounding extracellular matrix. These structures are called podosomes or invadopodia and collectively referred to as invadosomes. Due to their potential importance in both healthy physiology as well as in pathological conditions such as cancer, the characterization of these structures has been of increasing interest. Following early descriptions of invadopodia, assays were developed which labelled the matrix underneath metastatic cancer cells allowing for the assessment of invadopodia activity in motile cells. However, characterization of invadopodia using these methods has traditionally been done manually with time-consuming and potentially biased quantification methods, limiting the number of experiments and the quantity of data that can be analysed. We have developed a system to automate the segmentation, tracking and quantification of invadopodia in time-lapse fluorescence image sets at both the single invadopodia level and whole cell level. We rigorously tested the ability of the method to detect changes in invadopodia formation and dynamics through the use of well-characterized small molecule inhibitors, with known effects on invadopodia. Our results demonstrate the ability of this analysis method to quantify changes in invadopodia formation from live cell imaging data in a high throughput, automated manner.
References
[1]
Albiges-Rizo C, Destaing O, Fourcade B, Planus E, Block MR. 2009. Actin machinery and mechanosensitivity in invadopodia, podosomes and focal adhesions. Journal of Cell Science 122:3037-3049
[2]
Artym VV, Yamada KM, Mueller SC. 2009. ECM degradation assays for analyzing local cell invasion. Methods in Molecular Biology 522:211-219
[3]
Artym VV, Zhang Y, Seillier-Moiseiwitsch F, Yamada KM, Mueller SC. 2006. Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function. Cancer Research 66:3034-3043
[4]
Beaty BT, Sharma VP, Bravo-Cordero JJ, Simpson MA, Eddy RJ, Koleske AJ, Condeelis J. 2013. β1 integrin regulates Arg to promote invadopodial maturation and matrix degradation. Molecular Biology of the Cell 24:1661-1675
[5]
Block MR, Badowski C, Millon-Fremillon A, Bouvard D, Bouin AP, Faurobert E, Gerber-Scokaert D, Planus E, Albiges-Rizo C. 2008. Podosome-type adhesions and focal adhesions, so alike yet so different. European Journal of Cell Biology 87:491-506
[6]
Chan KT, Cortesio CL, Huttenlocher A. 2009. FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion. Journal of Cell Biology 185:357-370
[7]
Chen WT. 1989. Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells. Journal of Experimental Zoology 251:167-185
[8]
Destaing O, Block MR, Planus E, Albiges-Rizo C. 2011. Invadosome regulation by adhesion signaling. Current Opinion in Cell Biology 23:597-606
[9]
Haier J, Korb T, Hotz B, Spiegel HU, Senninger N. 2003. An intravital model to monitor steps of metastatic tumor cell adhesion within the hepatic microcirculation. Journal of Gastrointestinal Surgery 7:507-515
[10]
Hoshino D, Branch KM, Weaver AM. 2013. Signaling inputs to invadopodia and podosomes. Journal of Cell Science 126:2979-2989
[11]
Hoshino D, Jourquin J, Emmons SW, Miller T, Goldgof M, Costello K, Tyson DR, Brown B, Lu Y, Prasad NK, Zhang B, Mills GB, Yarbrough WG, Quaranta V, Seiki M, Weaver AM. 2012. Network analysis of the focal adhesion to invadopodia transition identifies a PI3K-PKCalpha invasive signaling axis. Science Signaling 5:ra66
[12]
Linder S, Wiesner C, Himmel M. 2011. Degrading devices: invadosomes in proteolytic cell invasion. Annual Review of Cell and Developmental Biology 27:185-211
[13]
Malpica N, de Solorzano CO, Vaquero JJ, Santos A, Vallcorba I, Garcia-Sagredo JM, del Pozo F. 1997. Applying watershed algorithms to the segmentation of clustered nuclei. Cytometry 28:289-297
[14]
Matov A, Applegate K, Kumar P, Thoma C, Krek W, Danuser G, Wittmann T. 2010. Analysis of microtubule dynamic instability using a plus-end growth marker. Nature Methods 7:761-768
[15]
Murphy DA, Courtneidge SA. 2011. The ‘ins’ and ‘outs’ of podosomes and invadopodia: characteristics, formation and function. Nature Reviews Molecular Cell Biology 12:413-426
[16]
Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M, Bradke F, Jenne D, Holak TA, Werb Z, Sixt M, Wedlich-Soldner R. 2008. Lifeact: a versatile marker to visualize F-actin. Nature Methods 5:605-607
[17]
Sharma VP, Entenberg D, Condeelis J. 2013a. High-resolution live-cell imaging and time-lapse microscopy of invadopodium dynamics and tracking analysis. Methods in Molecular Biology 1046:343-357
[18]
Sharma VP, Eddy R, Entenberg D, Kai M, Gertler Frank B, Condeelis J. 2013b. Tks5 and SHIP2 regulate invadopodium maturation, but not initiation, in breast carcinoma cells. Current Biology 23:2079-2089
[19]
Stoletov K, Strnadel J, Zardouzian E, Momiyama M, Park FD, Kelber JA, Pizzo DP, Hoffman R, VandenBerg SR, Klemke RL. 2013. Role of connexins in metastatic breast cancer and melanoma brain colonization. Journal of Cell Science 126:904-913
[20]
Tang H, Li A, Bi J, Veltman DM, Zech T, Spence HJ, Yu X, Timpson P, Insall RH, Frame MC, Machesky LM. 2013. Loss of Scar/WAVE complex promotes N-WASP- and FAK-dependent invasion. Current Biology 23:107-117
[21]
Tarone G, Cirillo D, Giancotti FG, Comoglio PM, Marchisio PC. 1985. Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes. Experimental Cell Research 159:141-157
[22]
Wang X, Fu X, Brown PD, Crimmin MJ, Hoffman RM. 1994. Matrix metalloproteinase inhibitor BB-94 (batimastat) inhibits human colon tumor growth and spread in a patient-like orthotopic model in nude mice. Cancer Research 54:4726-4728
[23]
Wang Y, McNiven MA. 2012. Invasive matrix degradation at focal adhesions occurs via protease recruitment by a FAK-p130Cas complex. Journal of Cell Biology 196:375-385
