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Application of Molecular Imaging to Breast Cancer: Imaging of CRIP1, a Gene Overexpressed in 90% of Human Breast Cancers.
Background. The sensitivity of screening technologies for early detection of breast cancer is limited and early detection of this cancer still presents a significant diagnostic challenge. Detection of cancer cells in a background of normal/hyperplastic benign tissue is often based on differences in physical properties between tissues, which are frequently minimal, i.e. small tumors are not detectable. To enhance detection of small tumors we are using genomic screening technologies to 1) identify the differences in molecular properties between cancer and normal tissues and 2) exploit these differences to increase the sensitivity of imaging for breast cancers.
Objective/Hypothesis. The underlying hypothesis for this work is that 1) a similar pattern of breast cancer-specific changes in gene expression are present in a high proportion of patients and that 2) the molecular differences between normal and cancer tissue can be used to significantly increase the sensitivity for detecting breast cancers non-invasively. We have in part demonstrated these hypothesizes by showing that the human transferrin receptor is up-regulated in a large proportion of breast cancers (41%) and that imaging probes targeting the receptor significantly increase the sensitivity of MR to detect small tumors (see attached manuscript). Here we propose a set of studies to design an imaging probe against a marker that is overexpressed in 90% of breast cancers.
Specific Aims. In collaboration with Dr. Sgroi (also a collaborator on this grant application) we have identified a potential molecular imaging marker for human breast cancer, cysteine rich intestinal peptide 1 (CRIP1). It is overexpressed over 8-fold in 90% of all tested human breast cancers and hyperplastic tissues (1) (n=63). We propose to develop molecular imaging agents against this target along the following aims:
Aim 1: Express and purify the CRIP1 protein, utilize phage display to identify high affinity peptides that bind CRIP1, and synthesize and characterize peptide binding.
Aim 2: Utilize Tat-peptide and other translocating peptides to traverse the cell membrane and image CRIP1 expressing cells in vitro.
Aim 3: Test in vivo the bio-distribution and imaging potential of the peptide using both optical and magnetic resonance imaging.
Study Design. To accomplish these goals CRIP1 cDNA will be cloned into a bacterial expression vector in frame with a histadine tag and enterokinase cleavage site for later purification. Following Cobalt column purification the protein will be cleaved and intact CRIP1 will be used for panning with a cys-7-cys phage library. Following several rounds of selection phage will be isolated and binding peptides identified. The peptides will then be characterized for their binding characteristics to CRIP1 both in vitro and in vivo.
Potential Outcomes and Benefits of the Research. Our preliminary work suggests that imaging the molecular rather than physical differences between tissues significantly increases the signal-to-noise ratio and allows detection of very small tumors (2, 3). This work has demonstrated how molecular imaging of cancer specific targets can substantially increase sensitivity of tumor detection. However, the target initially used was sub-optimal, only being overexpressed in 41% of breast cancers. Imaging based on CRIP1 will substantially increase the number of patients that will benefit from such a technology and the level of sensitivity for detection of breast cancers. This should result in greater cure rates for individuals afflicted with the disease. The initial studies proposed here will provide the groundwork for CRIP1-based non-invasive imaging of breast cancers and if successful should provide the necessary data for impetus toward developing these probes clinically.
Significantly, our ability to screen for early breast cancers is limited. While several early screening technologies for breast cancer detection have been employed, all have met with limited success. For example, probably the most prevalent screening procedure in Western counties, the mammogram, fails to detect approximately 30% of breast cancers in initial screenings. In general current imaging technologies for breast cancers utilize X-rays or sound waves to measure the differences in tissue density between tumors and surrounding tissues. Often times these differences are minimal and therefore small developing tumors are missed. In order to reduce the false negative rates, our laboratories have been trying to develop imaging technologies that do not depend on the physical differences between tissues to detect tumors. The underlying hypothesis for this work is that more sensitive detection of cancers will be achievable if we can utilize the underlying genetic changes in cancer for imaging. We have undertaken a non-conventional imaging program that utilizes genomic technologies to screen large groups of human breast cancer tissues for generalized changes in gene expression. In previous work performed with funding from the Komen Foundation (grant IMG00-000198), we demonstrated that it was possible to 1) use genomic screening technologies to identify the human transferrin receptor as an informative molecular marker for breast cancer and 2) develop molecular imaging probes that specifically detect the defined target non-invasively and in real time (see attached publications). These studies resulted in more sensitive detection of smaller tumors. Continuing our work initiated with Komen funding we have screened an increased number of human breast cancer tissues and have identified another potential molecular marker, CRIP1, that is specifically up-regulated in both early and later stages of breast cancer (up-regulated 8-fold or more in over 90% of tested breast cancers and hyperplastic tissues, n=63). This represents a much more pertinent imaging target with potential imaging application in all patients undergoing breast cancer screening. The research plan presented here, therefore, proposes to develop molecular imaging probes to target and detect this marker non-invasively, in vivo and in real time. If successful this work has the potential to develop probes that will allow earlier detection of developing breast cancers presumably resulting higher cure rates.