Enrichment and molecular characterization of single disseminated tumor cells (DTCs) and proof of circulating tumor cell (CTC)- and DTC-mediated tumorigenicity in vitro and in vivo
Final Report Abstract
In CRC as in most solid tumors, it is distant metastases rather than the primary tumor which limit the prognosis. However, CTCs are a highly heterogeneous tumor cell population, and only a small fraction of CTCs seem to be able to outgrow to clinically relevant tumors. In addition, colorectal-cancer derived CTCs are extremely rare, only a fraction of patients has detectable CTCs in the blood. This heterogeneity in combination with rarity results in tremendous technical and organizational obstacles for CTC research in CRC. In the second funding period, we aimed to culture CTCs, demonstrate their tumorigenicity and molecularly characterize disseminated tumor cells (DTCs), which are the dormant CTC subpopulation presumably found in the bone marrow. However, the above mentioned obstacles required us to redefine the goals of the funding period, as we were forced to recognize that the ambitious goals were not achievable with the resources at hand – many more patients along with substantially increased financial and staff resources would have been required. We therefore adapted the goals to the development of murine models for CTCs in order to further define the metastatic subpopulations of CTCs, and, as a second goal, aimed to determine whether colorectal liver metastases are able to shed viable and tumorigenic CTCs themselves. We were able to establish a orthotopic xenograft model of CRC which is able to reliably generate metastatic disease along with numerous CTCs. The molecular characterization of the CTCs revealed stem cell characteristics indicating the tumorigenicity of the CTCs; further experiments conclusively demonstrated the tumorigenicity of these CTCs both in vitro and in vivo. To our knowledge, this was the first experiment demonstrating colorectal CTCs to be tumorigenic. As a next step, we are currently evaluating a genetically engineered mouse model of CRC, which may be able to simulate the heterogeneity of colorectal CTCs more faithfully than the xenograft model. Initial results seem to be promising. In an attempt to determine whether hepatic metastases are able to metastasize themselves, we quantified CTCs in various blood compartments (central venous, hepatic venous, portal venous, arterial), isolated the CTCs and determined their phenotype. We found that if hepatic metastases are present, the number of CTCs in the hepatic venous blood compartment is much greater than in patients with disease only limited to the colon. The CTCs derived from hepatic metastases are viable and exhibit an invasive and stem-cell like phenotype, suggesting tumorigenicity. Interestingly, in patients with liver metastases (primary tumor resected), the number of CTCs in the portal vein is increased compared to patients without liver metastases and without primary tumor. This suggests that CTCs derived from liver metastases may be able to pass several capillary beds and remain in circulation, possibly contributing to intrahepatic recurrence after liver resection. In conclusion, while the original goals had to be adapted due to unexpected technical and biological obstacles, SP1 has been able to substantially add to the body of knowledge about the process of metastasis in CRC.
Publications
- Circulating tumor cells and prognosis of patients with resectable colorectal liver metastases or widespread metastatic colorectal cancer: a meta-analysis. Ann Surg Oncol 2013 20(7):2156-65
Groot Koerkamp B, Rahbari NN, Büchler MW, Koch M, Weitz J
(See online at https://doi.org/10.1245/s10434-013-2907-8) - Immune Escape and Survival Mechanisms in Circulating Tumor Cells of Colorectal Cancer. Cancer Res 2014;74:1694–704
Steinert G, Schölch S, Niemietz T, Iwata N, García SA, Behrens B, Voigt A, Kloor M, Benner A, Bork U, Rahbari NN, Büchler MW, Stoecklein NH, Weitz J, Koch M
(See online at https://doi.org/10.1158/0008-5472.CAN-13-1885) - Prognostic relevance of minimal residual disease in colorectal cancer. World J Gastroenterol 2014;20:10296–304
Bork U, Grützmann R, Rahbari NN, Schölch S, Distler M, Reissfelder C, Koch M, Weitz J
- Circulating tumour cells and outcome in non-metastatic colorectal cancer: a prospective study. Br J Cancer 2015;112:1306–13
Bork U, Rahbari NN, Schölch S, Reissfelder C, Kahlert C, Büchler MW, Weitz J, Koch M
(See online at https://doi.org/10.1038/bjc.2015.88) - Circulating tumor cells exhibit stem cell characteristics in an orthotopic mouse model of colorectal cancer. Oncotarget 2016;7:27232-42
Schölch S, García S, Iwata N, Niemietz T, Betzler A, Nanduri LK, Bork U, Kahlert C, Thepkaysone M, Swiersy A, Büchler MW, Reissfelder C, Weitz J, Rahbari NN
(See online at https://doi.org/10.18632/oncotarget.8373) - Metastatic Spread Emerging From Liver Metastases of Colorectal Cancer: Does the Seed Leave the Soil Again? Ann Surg 2016;263:345–52
Rahbari NN, Bork U, Schölch S, Reissfelder C, Thorlund K, Betzler A, Kahlert C, Schneider M, Ulrich AB, Büchler MW, Weitz J, Koch M
(See online at https://doi.org/10.1097/SLA.0000000000001341) - A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer. J Vis Exp 2017;125
Betzler AM, Kochall S, Blickensdörfer L, García SA, Thepkaysone M, Nanduri LK, Muders MH, Weitz J, Reissfelder C, Schölch S
(See online at https://doi.org/10.3791/55952) - Circulating Tumor Cells. Methods in Molecular Biology 1692:213-219, Humana Press (Springer, Berlin/Heidelberg/New York) 2017
García SA, Weitz J, Schölch S
(See online at https://doi.org/10.1007/978-1-4939-7144-2) - Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer. J Vis Exp 2017;125
Kochall S, Thepkaysone M, García SA, Betzler AM, Weitz J, Schölch S
(See online at https://doi.org/10.3791/55357)
