SP 1

Molecular characterization of single circulating and disseminated tumor cells

Results of the first funding period:
We successfully established a protocol for the isolation and genetic characterization of CRC patient-derived CTCs. After a first enrichment process with a density gradient centrifugation step contaminating CD45+ cells are depleted. The CTCs are ultimately identified by a fluorescently labeled anti-EpCAM antibody and manually picked with a micromanipulator as single cells. We were able to comprehensively characterize the genetic profile of CTCs and the associated tumor tissue. First, we performed an array-GCH-Assay on isolated CTCs and primary tumor. We could show that the isolated CTCs are genomically true tumor cells having similar chromosomal aberrations in comparison with tumor tissue. Once we established tumor cell identity, these cells were subsequently sequenced at single cell level and an expression profile of 47 genes responsible for stemness, proliferation, migration or invasion was created. CTCs were also screened for key mutations commonly exhibited in CRC, e.g. BRAF, KRAS or TP53. These mutations were found in an expected frequency for CRC. In more than 50% of patient samples CTCs and matched tumor did not show the same mutation profile. Gene expression analysis showed that epithelial markers as well as markers for migration or proliferation are down-regulated in CTCs. During circulation these cells are likely to be in a state of dormancy and consequently not accessible to the immune system. In summary, we could recognize a strong genetic heterogeneity between CTCs and corresponding primary tumor, and even between CTCs from the same patient.

Aims for the ongoing funding period:
1stDespite major advances in single-cell genomic analysis it is not possible to routinely characterize the expression profile of CRC-associated CTCs. Due to the fact that CTCs represent a fairly rare cell population and are only available by drawing blood from patients, it would be a further advancement and technical advantage to identify appropriate cell culture conditions to make CTCs cultivable.

2ndThe importance and relevance of CTCs research will be established by demonstrating tumorigenicity of these cells in in vitro as well as in in vivo models.

3rdIn the ongoing funding period, we aim for a comprehensive genetic characterization of disseminated tumor cells (DTCs) from human bone marrow samples.

Metastasis of epithelial carcinomas rather than primary tumor is the main cause for tumor related death. This process is highly sophisticated and requires the ability of tumor cells to enter the circulation, spread over the bloodstream or lymph system, invade target organs and subsequently form metastases. Circulating tumor cells (CTCs) are candidate cells that may be able to fulfill this sophisticated program. The prognostic significance of EpCAM+ CTCs in the blood of patients with colorectal cancer (CRC) has been convincingly shown in numerous studies. However, the molecular and especially the genetic aspects of these cells are still poorly understood.

Team SP1, from the left to the right: Dr. med. U. Bork, Dr. med. S. Schölch, Prof. Dr. med. J. Weitz, S. Ufer, Prof. Dr. med. M. Koch, M.D. S. Garcia, Dr. rer. nat. A. Kempe
Team SP 1

Project-related key publications

García SA, Swiersy A, Radhakrishnan P, Branchi V, Kanth Nanduri L, GyÅ Rffy B, Betzler AM, Bork U, Kahlert C, Reißfelder C, Rahbari NN, Weitz J, Schölch S. LDB1 overexpression is a negative prognostic factor in colorectal cancer. Oncotarget. 2016 Oct 5. doi: 10.18632/ oncotarget.12481. [Epub ahead of print]

Steinert G, Schölch S, Niemietz T, Iwata N, Garcia S, Behrens B, Voigt A, Kloor M, Benner A, Bork U,Rahbari NN, Büchler MW, Stoecklein N, Weitz J, Koch M. Immune Escape and Survival Mechanisms in Circulating Tumor Cells of Colorectal Cancer. Cancer Res. 2014;74(6):1694-1704.

Rahbari NN, Reissfelder C, Mühlbayer M, Weidmann K, Kahlert K, Büchler MW, Weitz J, Koch M. Correlation of circulating angiogenic factors with circulating tumor cells and disease recurrence in patients undergoing curative resection for colorectal liver metastases. Ann Surg Oncol 2011 Aug;18(8):2182-91. Epub 2011 May 20.

Rahbari NN, Bork U, Motschall E, Thorlund K, Büchler MW, Koch M, Weitz J. Molecular detection of tumor cells in regional lymph nodes is associated with disease recurrence and poor survival in node-negative colorectal cancer: a systematic review and meta-analysis. J Clin Oncol 2012 Jan 1;30(1):60-70. Epub 2011 Nov 28

Rahbari NN, Bork U, Kircher A, Nimitz T, Schölch S, Kahlert C, Schmidt T, Steinert G, Ulrich AB, Reissfelder C, Büchler MW, Koch M, Weitz J. Compartmental differences of circulating tumor cells in colorectal cancer. Ann Surg Oncol 2012 Jan 10. [Epub ahead of print].

Kahlert C, Lahes S, Radhakrishnan P, Dutta S, Mogler C, Herpel E, Brand K, Steinert G, Schneider M, Mollenhauer M, Reissfelder C, Klupp F, Fritzmann J, Wunder C, Benner A, Kloor M, Huth C, Contin P, Ulrich A, Koch M, Weitz J. Overexpression of ZEB2 at the invasion front of colorectal cancer is an independent prognostic marker and regulates tumor invasion in vitro. Clin Cancer Res 2011 Dec 15;17(24):7654-63. Epub 2011 Oct 31.

Kahlert C, Klupp F, Brand K, Lasitschka F, Diederichs S, Kirchberg J, Rahbari N, Dutta S, Bork U, Fritzmann J, Reissfelder C, Koch M, Weitz J. Invasion front-specific expression and prognostic significance of microRNA in colorectal liver metastases. Cancer Sci 2011 Oct;102(10):1799-807. Epub 2011 Aug 4

Antolovic D, Galindo L, Carstens A, Rahbari N, Büchler MW, Weitz J, Koch M. Heterogeneous detection of circulating tumor cells in patients with colorectal cancer by immunomagnetic enrichment using different EpCAM-specific antibodies. BMC Biotechnology 2010 Apr 28;10(1):35. [Epub ahead of print].

Rahbari NN, Aigner M, Thorlund K, Mollberg N, Motschall E, Jensen K, Diener MK, Büchler MW, Koch M, Weitz J. Meta-analysis shows that detection of circulating tumor cells indicates poor prognosis in patients with colorectal cancer. Gastroenterology 2010 May;138(5):1714-26. Epub 2010 Jan 25.

Lee YJ, Koch M, Karl D, Torres-Collado AX, Fernando NT, Rothrock C, Kuruppu D, Ryeom S, Iruela-Arispe ML, Yoon SS. Variable inhibition of thrombospondin 1 against liver and lung metastases through differential activation of metalloproteinase ADAMTS1. Cancer Res 2010 Feb 1;70(3):948-56. Epub 2010 Jan 26.

Fernando RT, Koch M, Rothrock C, Gollogly LK, D'Amore PA, Ryeom S, Yoon SS. Tumor escape from endogenous, extracellular matrix-associated angiogenesis inhibitors by up-regulation of multiple proangiogenic factors. Clin Cancer Res 2008 Mar 1;14(5):1529-39.

Koch M, Kienle P, Logan E, Antolovic D, Galindo L, Schmitz-Winnenthal FH, Schmidt J, Herfarth C, Weitz J. Detection of disseminated tumor cells in liver biopsies of colorectal cancer patients is not associated with a worse prognosis. Ann Surg Oncol 2007 Feb;14(2):810-7.

Koch M, Kienle P, Kastrati D, Antolovic D, Schmidt J, Herfarth C, von Knebel Doeberitz, Weitz J. Prognostic impact of hematogenous tumor cell dissemination in patients with stage II colorectal cancer. Int J Cancer 2006 Jun 15;118(12):3072-7.

Koch M, Kienle P, Hinz U, Antolovic D, Schmidt J, Herfarth C, von Knebel Doeberitz M, Weitz J. Detection of hematogenous tumor cell dissemination predicts tumor relapse in patients undergoing surgical resection of colorectal liver metastases. Ann Surg 2005 Feb;241(2):199-205.

Kienle P, Koch M, Autschbach F, Benner A, Treiber M, Wannenmacher M, von Knebel Doeberitz M, Büchler M, Herfarth C, Weitz J. Decreased detection rate of disseminated tumor cells of rectal cancer patients after preoperative chemoradiation: a first step towards a molecular surrogate marker for neoadjuvant treatment in colorectal cancer. Ann Surg 2003 Sep;238(3):324-30; discussion 330-1.

Weitz J, Koch M, Kienle P, Schrödel A, Willeke F, Benner A, Lehnert T, Herfarth C, von Knebel Doeberitz M. Detection of hematogenic tumor cell dissemination in patients undergoing resection of liver metastases of colorectal cancer. Ann Surg 2000 Jul;232(1):66-72.

Weitz J, Kienle P, Magener A, Koch M, Schrödel A, Willeke F, Autschbach F, Lacroix J, Lehnert T, Herfarth C, von Knebel Doeberitz M. Detection of disseminated colorectal cancer cells in lymph nodes, blood and bone marrow. Clin Cancer Res 1999 Jul;5(7):1830-6.

Weitz J, Kienle P, Lacroix J, Willeke F, Benner A, Lehnert T, Herfarth C, von Knebel Doeberitz M. Dissemination of tumor cells in patients undergoing surgery for colorectal cancer. Clin Cancer Res 1998 Feb;4(2):343-8.

Wong CW, Lee A, Shientag L, Yu J, Dong Y, Kao G, Al-Mehdi AB, Bernhard EJ, Muschel RJ. Apoptosis: an early event in metastatic inefficiency. Cancer Res 2001;61(1):333-8

Iinuma H, Okinaga K, Adachi M, Suda K, Sekine T, Sakagawa K, Baba Y, Tamura J, Kumagai H, Ida A. Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and K-ras genes in patients with colorectal cancer. Int J Cancer 2000 Jul 20;89(4):337-44.

Schmidt-Kittler O, Ragg T, Daskalakis A, Granzow M, Ahr A, Blankenstein TJ, Kaufmann M, Diebold J, Arnholdt H, Muller P, Bischoff J, Harich D, Schlimok G, Riethmuller G, Eils R, Klein CA. From latent disseminated cells to overt metastasis: Genetic analysis of systemic breast cancer progression. Proc Natl Acad Sci U S A 2003;100(13):7737-42.

Stoecklein NH, Hosch SB, Bezler M, Stern F, Hartmann CH, Vay C, Siegmund A, Scheunemann P, Schurr P, Knoefel WT, Verde PE, Reichelt U, Erbersdobler A, Grau R, Ullrich A, Izbicki JR, Klein CA. Direct genetic analysis of single disseminated cancer cells for prediction of outcome and therapy selection in esophageal cancer. Cancer Cell 2008 May;13(5):441-53.

Spaderna S, Schmalhofer O, Hlubek F, Jung A, Kirchner T, Brabletz T. Epithelial-mesenchymal and mesenchymal-epithelial transitions during cancer progression. Verh Dtsch Ges Pathol 2007;91:21-8.

Brabletz T, Hlubek F, Spaderna S, Schmalhofer O, Hiendlmeyer E, Jung A, Kirchner T. Invasion and metastasis in colorectal cancer: epithelial-mesenchymal transition, mesenchymal-epithelial transition, stem cells and beta-catenin. Cells Tissues Organs 2005;179(1-2):56-65.

Brabletz T, Jung A, Spaderna S, Hlubek F, Kirchner T. Opinion: migrating cancer stem cells - an integrated concept of malignant tumor progression. Nat Rev Cancer 2005;5(9):744-9.

O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumor growth in immunodeficient mice. Nature 2007;445(7123):106-10. Epub 2006 Nov 19.

Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R. Identification and expansion of human colon-cancer-initiating cells. Nature 2007;445(7123):111-5. Epub 2006 Nov 19.

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