Positron emission tomography (PET) allows one to measure tumor and normal tissue metabolism in vivo and non-invasively. We have been studying thymidine and its analogs, with the initial goal of imaging cellular proliferation. One of the analogs under study is FMAU {1-(2'-deoxy-2'-fluoro-beta-D- arabinofuranosyl)-thymine}, which we have labeled with 18F and studied in cell culture, animals and humans. Our studies found that in humans FMAU was readily retained in tumors, but not in normally proliferating bone marrow. Our work, and that of others, has demonstrated that FMAU is retained by the action of thymidine kinase 2 (TK2), a mitochondrial enzyme. We have found that increased retention reflects cellular stress. This is in contrast to 3'-deoxy-3'-fluorothymidine (FLT), which is retained by thymidine kinase 1 (TK1) the cytosolic enzyme associated with normal DNA synthesis. Here we will test the hypothesis that imaging cellular stress with 18F-FMAU may provide an early measure of cancer treatment response. Furthermore, FMAU may be simpler to use than agents being developed to image apoptosis, since the time course of stress may be more predictable than changes in apoptosis, which are often short term. We have already developed simplified approaches to 18F-FMAU imaging. To further understand and test 18F-FMAU for PET imaging we propose the following Aims.
Aim 1 will use cell cultures to determine how and when TK2 activity and FMAU uptake are controlled. We will extend our preliminary studies, which indicate that an altered, more active form of TK2 is generated during cellular stress. This will be analyzed using qRT-PCR to measure mRNA levels, Western blots to measure the protein and its isoforms, and enzymatic activity during the course of cisplatin treatment of lung cancer cell lines. Measurements of TK1, cell proliferation, apoptosis and mitochondrial mass will also be obtained for comparison with FMAU uptake.
Aim 2 will study the changes that occur after therapy using human lung cancer cell lines implanted in immunodeficient mice. Uptake of 18F-FMAU will be monitored with microPET to determine the best time for imaging after therapy. FMAU uptake will be compared to measures of cell proliferation, apoptosis, and TK2 expression from tumors removed at necropsy. Finally, Aim 3 will further study FMAU uptake in humans with lung cancer. We will study the reproducibility of FMAU imaging. We will measure FMAU uptake prior to resection of early stage disease and use the resected specimens to test the hypothesis that FMAU uptake correlates with tumor TK2 activity. We will also measure proliferation, TK1, and apoptosis.
This Aim will then study the time course of FMAU retention after treatment in patients receiving standard chemotherapy for lung cancer. This will test the hypothesis that early increases in FMAU uptake reflect cellular stress and ultimate response to therapy. In summary, our studies seek to validate and test the use of 18F-FMAU in cell cultures, mice and humans, as a marker of cellular stress which reflects TK2 activity and predicts treatment response.
The studies being proposed will result in methods to measure tumor activity and treatment-induced changes within hours to days after the start of therapy. Currently there is no routine proven way of doing this early in the course of treatment. If this approach is validated it will be very useful in tailoring therapy that is more successful in killing tumors. While we have chosen to study lung cancer here, FMAU may well find use in the evaluation of other tumors as well. We conjecture that validation of its use in lung tumors will expedite its use with other cancers.
