Novel models of metastasis must be developed to initiate new therapeutic approaches, since metastases from colorectal carcinoma (CRC) still kill 45% of patients. In the parent R01 grant CRC cells injected intrasplenically into athymic nude mice is a useful model because growth in liver is significantly associated with prognosis in patients. However, this in vivo model is expensive and cumbersome. We have now developed an in vitro organotypic culture system that uses a NASA-designed rotating wall vessel (RWV) bioreactor to promote cell and tissue colocalization with low shear stress. The RWV approximates solid body rotation by rotating cells and tissues in aqueous medium around the horizontal axis. Normal mouse liver fragments and cells are viable in the RWV for at least 8-10 days. Using the RWV with ischemic mouse liver reoxygenated in vitro and co-cultured with CRC, we have recreated the survival kinetics of CRC in the first 24 hr. In this supplemental application we propose to extend this in vitro model from 24 hr to 7 days and determine whether it can be used to assess the importance of host cytokine and growth factor interactions. Our overall hypothesis is that organotypic culture in an RWV recreates the host-tumor microenvironment during the first week of metastasis with sufficient fidelity to predict the ability of human CRC to colonize liver in vivo. Our secondary postulate is that carcinoembryonic antigen (CEA), IL-10, IL-6 and/or pleiotrophin are essential growth factors for human CRC metastasis.
Our specific aims are to: 1.) Establish the kinetics of cell number, viability, and S phase fraction in vivo after CX-1 or Clone A cells are injected intrasplenically; 2.) Determine the optimal conditions for murine liver fragment:CRC cell co-culture to best approximate the proliferation of CRC cells in vivo in the liver after intrasplenic injection; 3.) Determine whether CEA and the cytokines that it induces, IL-6 and IL-10, effect CRC cell proliferation in the RWV co-cultures after the first 24 hr; and 4.) Determine whether pleiotrophin (PTN) is an essential growth factor for CRC metastasis in vivo and the RWV in vitro. Our overall purpose is to extend the original R01 into a more robust model of the first 7 days of liver colonization and to develop an organotypic model that uses fewer mice but facilitates preclinical testing of new therapies for metastasis.
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