Abstract 977: Primary colon cancer tumor initiating cells with mesenchymal phenotype initiate metastases and hibernate in the bone marrow in immunodeficient mice
Authors: Hoffmann Christopher M. , Ball Claudia R. , Dieter Sebastian , Nowrouzi Ali , Brand Karsten , Koch Moritz , Schmidt Manfred , Weitz Juergen , von Kalle Christof , Glimm Hanno 
Published: June 2012

Abstract
Colorectal cancer is one of the most common cancer related causes of death in the western world. In most cases, patients do not die due to primary tumor but to metastases formation in distant organs. Recent evidence indicates that a small subset of cells in the primary tumor undergoes a process called epithelial to mesenchymal transition (EMT), which enables these cells to leave the primary site, enter the vasculature and survive in the blood. Moreover, the process of EMT can be reversed to initiate a metastasis resembling the primary lesion. The underlying mechanisms that drive EMT and allow disseminated cells to survive in the blood remain to be identified.
To address this question, we cultured primary colorectal cancer cells derived from 9 different patients as tumor spheres under serum-free conditions and transplanted these sphere cells under the kidney capsule of immunocompromised NOD/SCID-IL2RGnull (NSG) mice. In all cases, 5*104 cells from the primary sphere cultures where sufficient to initiate tumors. Interestingly, we observed frequent metastasis formation into liver or lung of transplanted mice in 3 of 9 patient samples. Compared to non-metastasizing primary sphere cultures, metastasizing spheres strongly expressed the mesenchymal marker vimentin and down regulated the epithelial markers E-cadherin and several keratins. To analyze the clonal origin of metastasis initiating cells we lentivirally marked sphere cells prior to transplantation using an eGFP expressing lentiviral vector. Highly sensitive tracking of individual clones by LAM-PCR on DNA-samples obtained from transplanted mice demonstrated that all clones within metastases were also present in the initial tumor under the kidney capsule. This indicates that transplanted cells first initiated tumor formation and subsequently metastasized. Moreover, a considerable number of clones were detected in the bone marrow of mice with distant metastases, while only very few clones were found in the peripheral blood. Serial transplantation of bone marrow derived cells from mice carrying metastases led to tumor formation, demonstrating that human colon cancer tumor initiating cells enter the circulation only transiently but persist in the bone marrow. These results identify the bone marrow as an important niche for tumor-initiating cell hibernation.
In summary, we demonstrate that only primary colon cancer cells highly expressing EMT-associated genes have the potential to leave the primary lesion, survive in the blood, hibernate in the bone marrow and initiate metastases in a xenotransplantation model. This model allows to dissect the the mechanisms of EMT and metastases formation in human colon cancer and to evaluate novel targeted therapies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 977. doi:10.1158/1538-7445.AM2011-977