Specific Aims- The goal of this project is to develop non-invasive molecular imaging metrics to assess treatment response to targeted therapeutics in colorectal cancer (CRC). Currently, imaging criteria for evaluating therapeutic efficacy is based upon anatomical information according to Response Evaluation Criteria in Solid Tumors (RECIST) guidelines (Therasse, Arbuck et al. 2000). These criteria, which are solely based on a reduction in tumor size, do not take advantage of the cellular and molecular information now available through novel imaging methods (Jaffe 2006). Furthermore, even though cellular and molecular changes may occur within hours of treatment, RECIST criteria and associated imaging methods are frequently inadequate for assessing early tumor response because changes in tumor size may require weeks/months of treatment. Therefore, given these limitations, there is a tremendous need to develop novel imaging metrics that can assess cellular and molecular responses of tumor cells to therapy at the earliest possible time. In this project, we will integrate both pre-clinical and clinical data accessible through our participation in the Gastrointestinal Special Program of Research Excellence (GI SPORE) and in the Mouse Models of Human Cancers Consortium (MMHCC), to determine the utility of novel molecular imaging techniques to provide relevant, quantitative readouts of therapeutic efficacy. Both the GI SPORE and the Vanderbilt MMHCC are large, multi-investigator, NCI-funded programs focused on CRC. Our involvement in these programs enables us to address important problems bridging cellular and molecular biology and molecular imaging. As such, we propose to develop, characterize, and validate molecular imaging metrics to assess tumor cell proliferation, tumor cell apoptosis, and EGFR and VEGR expression, non-invasively To characterize these in viva imaging metrics, we will follow the cellular and molecular response of colon cancer cells to therapies targeting EGFR and SRC activity, first as single agent regimens, and then as combined regimens. ? In Aim 1 we will image cellular response to a well characterized and clinically active EGFR-targeted monoclonal antibody (cetuximab; also known as Erbitux(r) and C225) that has demonstrated efficacy in CRC, as well as two small-molecule tyrosine kinase inhibitors (TKIs), one directed against EGFR tyrosine kinase (EKl-785) and the other against SRC tyrosine kinase (dasatinib).
In Aim 2, we will image similar measures of tumor cell response to combined blockade of two components of the EGFR axis using C225 and EKI-785, as well as to combined blockade of two complementary pathways, EGFR and SRC kinase using C225 and desatinib. Additionally, Aim 2 will also test combination therapies in a hepatic metastasis mouse model of CRC in order to image tumor cell responses that may be dependent on tumor microenvironment. Throughout the study, non-invasive imaging metrics will be validated against tumors excised from treated and untreated animals by quantitative molecular techniques including histopathology qRT-PCR, mass spectrometry and western blot analysis, to correlate the imaging readouts with cellular and molecular events occurring in response to therapy. ? ? AIM 1) To non-invasively assess the effects of EGFR- and SRC-targeted therapies on human colorectal tumor xenografts on critical biological processes (proliferation, apoptosis, angiogenesis, EGFR expression and overall metabolism). Baseline [18F]-FLT-PET (proliferation), NIR700-Annexin V (apoptosis), NIR800-aVEGFR2 (angiogenesis), NIR800-EGF (EGFR) and [18F]-FDG-PET (metabolism) imaging will be performed and will be repeated after 3 doses of an EGFR monoclonal antibody (cetuximab), an irreversible EGFR tyrosine kinase inhibitor (EKI-785) and a SRC tyrosine kinase inhibitor (SKI; dasatinib) as monotherapy for two human colorectal cancer cell lines, DiFi and HCT-116. The RECIST-based responses have been defined for these agents; however, information regarding the in viva cellular and molecular responses for these agents are lacking. In this aim, the non-invasive imaging parameters named above will be validated by molecular analysis of the corresponding biological processes (Kl-67 [proliferation] caspase 3 [apoptosis], CD31 and CD309 [angiogenesis], total and phospho-EGFR, total and phospho-FAK paxillin and p130CAS [SRC activity]) in normal (colon and liver) and neoplastic tissues harvested within one hour from completion of the last imaging session. Validation of these imaging methods in mouse models fulfills a necessary step prior to application of these methods to clinical studies including ongoing clinical trials. ? ? AIM 2) To assess a combined EGFR blockade with cetuximab and EKI-785 compared to combined EGFR and SRC blockade with cetuximab and dasatinib using non-invasive imaging of DiFi and HCT-116 xenografts and an HCT-116 liver metastasis model of colorectal cancer. We will directly compare the cellular and molecular responses of DiFi and HCT-116 flank injected tumors and HCT-116 liver metastases to targeted therapeutics in viva. For these, studies, we will deliver combined therapeutics of cetuximab and EKI-785 or cetuximab and dasatinib. A low response rate to EGFR blockade therapeutics has been modeled by the acquisition of compensatory mechanisms of resistance or collaborating signal transduction pathways that allow tumor cells to escape the blockade, and may even act to sustain the cancer in different ways. In response to such potential for non-overlapping mechanisms of drug resistance, this specific aim will study the combined pharmacological blockade of EGFR and SRC. Based on preliminary data generated in our laboratory, we believe that these combinations represent promising strategies to improve the treatment of human metastatic CRC and may serve as a paradigm for the treatment of other solid neoplasms. To carry out this task, we propose a combination of pre-clinical studies in a conventional xenograft mouse models of CRC as well as in a mouse model of hepatic metastasis. ? ? The overarching goals of this project are to test the reliability of molecularly-targeted imaging metrics for assessing tumor cell biology within the context of targeted therapeutics, and to examine the role that tumor microenvironment plays in tumor cell response. This project integrates with the overall goals of ICMIC in two fundamental ways: (1) it addresses a significant need to update conventionally accepted RECIST-based response criteria and (2) it addresses a need to develop standardized high-throughput preclinical models for therapeutic testing. As added benefit, this project proposes to develop non-invasive metrics to assess treatment response in CRC to the action of promising drugs currently being evaluated in clinical trials. ? ? Thus, we envision that results obtained in these investigations have direct translational potential. Furthermore, we anticipate that findings from this study will be of high biological relevance, particularly since our studies include characterization of the proposed imaging metrics within the context of a relevant tumor microenvironment. Going forward, we envision that these findings will be applied towards ongoing clinical trials testing EGFR and SRC-targeted therapies, with additional potential for broad application to other targeted therapeutic regimens and tumor types. ? ? ? ?
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