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(12) United States Patent (10) Patent No.: US 8,298,756 B2 Condeelis (45) Date of Patent: Oct

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(12) United States Patent (10) Patent No.: US 8,298,756 B2 Condeelis (45) Date of Patent: Oct US008298756B2 (12) United States Patent (10) Patent No.: US 8,298,756 B2 Condeelis (45) Date of Patent: Oct. 30, 2012 (54) ISOLATION, GENE EXPRESSION, AND Jang et al (Clinical and Experimental Metastasis, 1997, vol. 15, pp. CHEMOTHERAPEUTC RESISTANCE OF 469-483).* MOTLE CANCER CELLS Wyckoff JB et al., entitled “The Collection of the Motile Population of Cells from a Living Tumor.” Cancer Research 60:5401-5404, Oct. (75) Inventor: John S. Condeelis, City Island, NY (US) 2000. Wang W et al., Gene expression analysis on Small numbers of (73) Assignee: Albert Einstein College of Medicine of invasive cells collected by chemotaxis from primary mammary Yeshiva University, Bronx, NY (US) tumors of the mouse, BMC Biotechnology 3:13, Aug. 2003, 12 pageS. Wang Wet al., entitled "Single cell behavior in metastatic primary (*) Notice: Subject to any disclaimer, the term of this mammary tumors correlated with gene expression patterns revealed patent is extended or adjusted under 35 by molecular profiling.” Cancer Research 62: 6278-6288, Nov. 2002. U.S.C. 154(b) by 318 days. Santala Met al., entitled “Synthesis and breakdown of fibrillar col lagens: concomitant phenomena in ovarian cancer. British Journal (21) Appl. No.: 11/659,514 of Cancer 77(11):1825-1831, 1998, Abstract Only. LiT et al., entitled "Inhibiting Ras signaling in the therapy of breast (22) PCT Filed: Aug. 4, 2005 cancer.” Cinical Breast Cancer 3(6):405-416, 2003, Abstract Only. Thigpen JT, entitled "Chemotherapy for advanced ovarian cancer: (86). PCT No.: PCT/US2005/027680 overview of randomized trails.” Semin. Oncol. 27(3 Suppl7): 11-16, 2000, Abstract Only. S371 (c)(1), Supplementary European Search Reportdated Jun. 2, 2008 for Appli (2), (4) Date: Sep. 26, 2007 cation No. EP 05807467.5, 2 pages. Goswami S. et al., entitled “Breast Cancer Cells Isolated by (87) PCT Pub. No.: WO2006/017635 Chemotaxis from Primary Tumors Show Increased Survival and Resistance to Chemotherapy,” Cancer Research, Nov. 1, 2004, vol. PCT Pub. Date: Feb. 16, 2006 64, pp. 7664-7667. Santala Met al., entitled “Synthesis and breakdown of fibrillar col (65) Prior Publication Data lagens: concomitant phenomena in ovarian cancer. British Journal US 2008/O138805 A1 Jun. 12, 2008 of Cancer (1998) 77(11), 1825-1831. Thigpen JT, entitled “Chemotherapy for Advanced Ovarian Cancer. Related U.S. Application Data Overview of Randomized Trials.” Seminars in Oncology, vol. 27. No. 3, Suppl 7 Jun. 2000, 11-16. (60) Provisional application No. 60/600,697, filed on Aug. LiT et al., entitled "Inhibiting Ras Signaling in the Therapy of Breast 11, 2004. Cancer.” Clinical Breast Cancer, Feb. 2003, 405-416. (51) Int. Cl. * cited by examiner CI2O I/68 (2006.01) Primary Examiner — Karen Canella GOIN33/574 (2006.01) (74) Attorney, Agent, or Firm — Amster, Rothstein & (52) U.S. Cl. ......... 435/4; 435/6.14; 435/6.17; 435/723; Ebenstein LLP 435/7.24 (58) Field of Classification Search ........................ None (57) ABSTRACT See application file for complete search history. Methods of isolating motile cells of interest from an animal (56) References Cited tissue is provided. Also provided are methods of determining mRNA or protein expression of a gene in motile cells of U.S. PATENT DOCUMENTS interest from an animal tissue. Additionally, methods of deter 2005/0014208 A1 1/2005 Krehan et al. ............... 435/723 mining whether a cancer in a tissue of a mammal is likely to metastasize are provided. Methods are also provided for FOREIGN PATENT DOCUMENTS inhibiting metastasis of a cancer in a tissue of a mammal. WO WOO3,O23060 * 3/2003 Further provided are methods of determining resistance of a WO WO2004/O15396 * 2/2004 motile cancer cell population in an animal tissue to a chemo OTHER PUBLICATIONS therapeutic agent. Dong et al (Cancer Research, 2001, vol. 61, pp. 4797-4808).* 14 Claims, 11 Drawing Sheets U.S. Patent Oct. 30, 2012 Sheet 1 of 11 US 8,298,756 B2 FIG. 1 . U.S. Patent Oct. 30, 2012 Sheet 2 of 11 US 8,298,756 B2 FG. 2 F. Anti-apoptotic genes Description le 3 immediate early response 3 Ubl 1a2 ubiquitin-like 1 (sentrin) activating enzyme subunit 2 TXI Hsp105 heat shock protein, 105 kDa Odc ornithine decarboxylase, structural 4. Dad Trp53 2 5 Hsp60 A. i4 P Cldn3 Apis o Ap il apoptosis inhibitor 1 o Adam17 2 O Pdcd4 rogrammed cell death 4 Femb eminization 1 b homolog (C. elegans) f Apaf1 apoptotic protease activating factor 1 Pdcd8 programmed cell death 8 (apoptosis inducing factor) Cellular apoptosis susceptibility protein ESTs. Highly similar to apoptosiss Apoptosis-inducing factor AlF D 4.5 ra B 4.) 3.5 3D 20 RT-PCR 1.5 1) .5 0. Apia Brca2 Dad 1 Flsp60 Itabi if Od Genes tested U.S. Patent Oct. 30, 2012 Sheet 3 of 11 US 8,298,756 B2 FIG. 3 S83 eVEO3e3 at 0. U.S. Patent Oct. 30, 2012 Sheet 4 of 11 US 8,298,756 B2 F.G. 4 / A?traeaeaer, U.S. Patent Oct. 30, 2012 Sheet 5 of 11 US 8,298,756 B2 cDNA microarray profiling-gene Neele collection expression of of it wasive cells invasive cells (i) FACS churciulon a general population of cells from pittay carcinoma cells (ii) to' cDNA microarray profiling-gene Marigel expression altered by: Mathigel, EGF EGF.Matrigel (iv) application U.S. Patent Oct. 30, 2012 Sheet 6 of 11 US 8,298,756 B2 F.G. 6 Annotated genes ESS 1. Cell Cycle F.Cytoskeletan & ECM 2. Apoptosis 8.Membrane associated O3. Tumor suppressor & Oncogenes 9.Growth factors & Signal Transduction 4. Development & Differentiation 0.ucleic acid chemistry 5. Metabolisrn 1.Others E. S. Protein fetablish U.S. Patent US 8,298,756 B2 Ioun Aleuci pe3elop speeN U.S. Patent US 8,298,756 B2 U.S. Patent Oct. 30, 2012 Sheet 9 of 11 US 8,298,756 B2 FIG. 8 U.S. Patent Oct. 30, 2012 Sheet 10 of 11 US 8,298,756 B2 FG, 9 A us EGF(N) it as Siano Eof. E25nM EGF U.S. Patent Oct. 30, 2012 Sheet 11 of 11 US 8,298,756 B2 F.G. 10 Bigot once T3-1 -21 s 2 s i Tugnor growth s f A321 Ln31 US 8,298,756 B2 1. 2 ISOLATION, GENE EXPRESSION, AND Clark, E. A., Golub, T. R. Lander, E. S., and Hynes, R. O. CHEMOTHERAPEUTC RESISTANCE OF Genomic analysis of metastasis reveals an essential role for MOTILE CANCER CELLS RhoC. Nature, 406: 532-535, 2000. Condeelis, J. and Segall, J. E. Intravital imaging of cell move CROSS-REFERENCE TO RELATED ment in tumours. Nat Rev Cancer, 3: 921-930, 2003. APPLICATION Condeelis, J., Song. X., Backer, J., Wyckoff, J., and Segall, J. Chemotaxis of cancer cells during invasion and metastasis. This is a U.S. national phase of PCT Application No. In: 5th Abercrombie Symposium on Cell Behaviour, St. PCT/US2005/027680 filed Aug. 4, 2005, which claims the Catherine's College, Oxford, UK, 2003. benefit of U.S. Provisional Application No. 60/600,697, filed 10 Cooper, J.A. and Schafer, D. A. Control of actin assembly and Aug. 11, 2004. disassembly at filament ends. Curr Opin Cell Biol, 12: 97-103, 2000. STATEMENT OF GOVERNMENT SUPPORT Coulombe, P. A. and Omary, M. B. Hard and soft prin 15 ciples defining the structure, function and regulation of This invention was made with government Support under keratin intermediate filaments. Curr Opin Cell Biol, 14: grant numbers CA089829 and CA 100324 awarded by the 110-122, 2002. National Institutes of Health. The government has certain Dal Canto, R.A., Shaw, M.K., Nolan, G.P., Steinman, L., and rights in the invention. Fathman, C. G. Local delivery of TNF by retrovirus-trans duced T lymphocytes exacerbates experimental autoim BACKGROUND mune encephalomyelitis. Clin Immunol, 90:10-14, 1999. Davila, M., Frost, A. R., Grizzle, W. E., and Chakrabarti, R. (1) Field of the Invention LIM Kinase 1 Is Essential for the Invasive Growth of The present invention generally relates to the characteriza Prostate Epithelial Cells: IMPLICATIONS IN PROSTATE tion of motile cells and invasive cells of tumors. More spe 25 CANCER. J. Biol Chem, 278: 36868-36875, 2003. cifically, the invention is directed to methods of isolating Edwards, D.C., Sanders, L. C., Bokoch, G. M., and Gill, G. motile cells, in particular invasive cells, and the characteriza N. Activation of LIM-kinase by Pak1 couples Rac/Cdc42 tion of gene expression in those cells. GTPase signalling to actin cytoskeletal dynamics. Nat Cell (2) Description of the Related Art Biol, 1:253-259, 1999. References Cited 30 Evan, G. I. and Vousden, K. H. Proliferation, cell cycle and Awada, A., Cardoso, F., Atalay, G., Giuliani, R., Mano, M., apoptosis in cancer. Nature, 411: 342-348, 2001. and Piccart, M.J. The pipeline of new anticancer agents for Farina, A.R., Coppa, A., Tiberio, A., Tacconelli, A., Turco, A., breast cancer treatment in 2003. Crit Rev. Oncol Hematol, Colletta, G., Gulino, A., and Mackay, A. R. Transforming 48: 45-63, 2003. growth factor-betal enhances the invasiveness of human Bailly, M. and Condeelis, J. Cell motility: insights from the 35 MDA-MB-231 breast cancer cells by up-regulating uroki backstage. Nat Cell Biol. 4: E292-294, 2002. nase activity. IntJ Cancer, 75: 721-730, 1998a. Bailly, M., Ichetovkin, I., Grant, W., Zebda, N., Machesky, L. Farina, K. L., Wyckoff, J. B., Rivera, J., Lee, H., Segall, J. E., M., Segall, J. E., and Condeelis, J. The F-actin side binding Condeelis, J. S., and Jones, J. G. Cell motility of tumor activity of the Arp2/3 complex is essential for actin nucle cells visualized in living intact primary tumors using green ation and lamellipod extension.
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