Optical coherence tomography and targeted multi-modal protein microspheres for cancer imaging

The field of biomedical optics has grown quickly over the last two decades as various technological advances have helped increase the acquisition speeds and the sensitivity limits of the technology. During this time, optical coherence tomography (OCT) has been explored for a wide number of clinical applications ranging from cardiology to oncology to primary care. In this thesis, I describe the design and construction of an intraoperative clinical OCT system that can be used to image and classify breast cancer tumor margins as normal, close, or positive. I also demonstrate that normal lymph nodes can be distinguished from reactive or metastatic lymph nodes by looking at the difference in scattering intensity between the cortex and the capsule of the node. Despite the advances of OCT in the detection and diagnosis of breast cancer, this technology is still limited by its field of view and can only provide structural information about the tissue. Structural OCT would benefit from added contrast via sub-cellular or biochemical components via the use of contrast agents and functional OCT modalities. As with most other optical imaging techniques, there is a trade off between the imaging field of view and the high-resolution microscopic imaging. In this thesis, I demonstrate for the first time that MM-OCT can be used as a complimentary technique to wide field imaging modalities, such as magnetic resonance imaging (MRI) or fluorescence imaging, using targeted multi-modal protein microspheres. By using a single contrast agent to bridge the wide field and microscopic imaging modalities, a wide field imaging technique can be used to initially localize the contrast agent at the site of interest to guide the location of the MM-OCT imaging to provide a microscopic view. In addition to multi-modal contrast, the microspheres were functionalized with RGD peptides that can target various cancer cell lines. The cancer cells readily endocytosed bound protein microspheres, revealing the possibility that these protein microspheres could also be used as therapeutic agents. These investigations furthered the utility of the OCT technology for cancer imaging and diagnosis.

Targeted multi-modal protein microspheres for cancer imaging

Optical coherence tomography (OCT) is a novel technology that has been developed for various clinical applications from ophthalmology to oncology. OCT is analogous to ultrasound but with micron-scale resolution by using light waves instead of sound waves to provide detailed structural information at the cellular level. The development of contrast agents has been critical to the development of OCT and its functional modalities such as magneto-motive OCT (MM-OCT). MM-OCT is a modality of OCT in which a small external magnetic field is modulated on and off during imaging, providing an added dimension of contrast from the magnetic particle responses. Protein microspheres consisting of a hydrophobic oil core and a hydrophilic BSA protein shell provide the vehicle for a multi-modal contrast agent. The microspheres encapsulate iron oxide nanoparticles in the oil core, providing magnetic signal contrast, and dyes such as Nile Red and DiR iodide, providing fluorescence contrast. The outer surface is functionalized using a layer-by-layer adhesion process to attach RGD peptide sequences to target integrin receptors. Using dynamic light scattering, we found the size distribution of the microspheres to be between 1-5 µm. Under SEM and TEM, we were able to visualize the various layers and coatings, such as silica and RGD peptides, of the microsphere. The microspheres were optimized to maximize the magnetic contrast under MM-OCT and MRI, and the fluorescent contrast under a dark box fluorescence imaging system, and fluorescence microscopy. These studies validated the use of MM-OCT as a method for quantifying the relative amount of iron oxide and the relative number of microspheres in the samples. To address the binding specificity and sensitivity of the RGD coated microspheres to the integrin receptors, the microspheres were incubated with cell lines of varying expression levels of the alpha(v)beta(3) integrin receptor and visualized under fluorescence microscopy. The cell lines used in this study included a normal epithelial cell line: hTERT-HME1, and several human breast cancer cell lines: HCC38, SK-BR-3, MCF-7, ZR-75-1, MDA-MB-231, and MDA-MB-435S. These results were externally validated by quantification of the receptors using indirect immunohistochemical staining and flow cytometry. Preliminary results, using the multi-spectral dark box fluorescence imaging system, demonstrate the localization of the microspheres to the vasculature surrounding the tumor and to lymph nodes. This is highly suggestive of the microsphere’s selective binding to the vasculature. By combining the benefits of these various imaging modalities in a single agent, it becomes possible to use a wide-field imaging method such as MRI or small animal fluorescence imaging to initially locate the agents in-vivo, to use MM-OCT to provide micron scale resolution structural images in-vivo, and to use fluorescence microcopy to confirm the localization of these particles ex-vivo.


Targeted multifunctional multimodal protein-shell microspheres as cancer imaging contrast agents

PURPOSE: In this study, protein-shell microspheres filled with a suspension of iron oxide nanoparticles in oil are demonstrated as multimodal contrast agents in magnetic resonance imaging (MRI), magnetomotive optical coherence tomography (MM-OCT), and ultrasound imaging. The development, characterization, and use of multifunctional multimodal microspheres are described for targeted contrast and therapeutic applications.PROCEDURES: A preclinical rat model was used to demonstrate the feasibility of the multimodal multifunctional microspheres as contrast agents in ultrasound, MM-OCT and MRI. Microspheres were functionalized with the RGD peptide ligand, which is targeted to α(v)β₃ integrin receptors that are over-expressed in tumors and atherosclerotic lesions.RESULTS: These microspheres, which contain iron oxide nanoparticles in their cores, can be modulated externally using a magnetic field to create dynamic contrast in MM-OCT. With the presence of iron oxide nanoparticles, these agents also show significant negative T2 contrast in MRI. Using ultrasound B-mode imaging at a frequency of 30 MHz, a marked enhancement of scatter intensity from in vivo rat mammary tumor tissue was observed for these targeted protein microspheres.CONCLUSIONS: Preliminary results demonstrate multimodal contrast-enhanced imaging of these functionalized microsphere agents with MRI, MM-OCT, ultrasound imaging, and fluorescence microscopy, including in vivo tracking of the dynamics of these microspheres in real-time using a high-frequency ultrasound imaging system. These targeted oil-filled protein microspheres with the capacity for high drug-delivery loads offer the potential for local delivery of lipophilic drugs under image guidance.


Fourier Transform Light Scattering (FTLS) of Cells and Tissues

Fourier transform light scattering (FTLS) has been recently developed as a novel, ultrasensitive method for studying light scattering from inhomogeneous and dynamic structures. FTLS relies on quantifying the optical phase and amplitude associated with a coherent image field and propagating it numerically to the scattering plane. In this paper, we review the principle and applications of FTLS to static and dynamic light scattering from biological tissues and live cells. Compared with other existing light scattering techniques, FTLS has significant benefits of high sensitivity, speed, and angular resolution. We anticipate that FTLS will set the basis for disease diagnosis based on intrinsic tissue optical properties and provide an efficient tool for quantifying cell structures and dynamics.


Congressionally Directed Medical Research Programs – Breast Cancer Research Program

Freddy Nguyen, an M.D./Ph.D. student in Professor Stephen Boppart’s Biophotonics Imaging Laboratory, was awarded an FY07 BCRP
Predoctoral Traineeship Award to optimize the use of an innovative imaging technology, magnetomotive optical coherence tomography (MM-OCT), which can provide real-time microscopic analysis of tumor
cells. Specifically, Mr. Nguyen’s project is to develop and optimize protein microspheres as a multimodal contrast agent to be used in conjunction with MM-OCT.
Mr. Nguyen has focused on encapsulating iron oxide nanoparticles and fluorescent dyes into the inner cores of modified protein microspheres capable of specifically targeting tumor neovessels, which are the blood vessels that tumors form to support their rapid growth. Tumor neovessel specificity was achieved by coating the microspheres with an arginine-glycine-asparatate (RGD) peptide, which binds to the αvβ3 integrin receptor on the surface of tumor neovessel endothelial cells. Preliminary studies confirmed that the microspheres preferentially bind to the tumor cells because they overexpress αvβ3 integrins in vitro. The microspheres accumulated in the neoves- sels at the tumor sites when injected into tumor-bearing rats. Mr. Nguyen plans to further pur- sue the cancer-specific targeting of the protein microspheres as a potential diagnostic contrast agent as well as a therapeutic agent in the treatment of breast cancer.

RGD coated protein microspheres as a dual fluorescent and magnetomotive contrast agent for targeted cancer imaging with magnetomotive optical coherence tomography

Optical coherence tomography (OCT) is a novel technology that has been developed for various clinical applications ranging from ophthalmology to oncology. OCT is analogous to ultrasound technology but with micron by using light waves instead of sound waves providing detailed morphological or structural information at the cellular level about the tissue specimen. Magneto-motive OCT (MM-OCT) is a recently developed modality of OCT in which a magnetic field is modulated on and off during imaging. With the development of this modality, exogeneous contrast agents are becoming more important to target markers that are expressed prior to morphological changes that structural OCT can only detect. Modified protein microspheres consisting of an oil core and a hydrophilic BSA protein shell are being presented as a multi-modal contrast agent vehicle. The protein microspheres are encapsulated with iron oxide in the oil core to provide the magnetic signal contrast and a near infrared dye to provide a fluorescence contrast. The outer surface is functionalized using a layer-by-layer adhesion process to attach RGD peptide sequences to target integrin receptors. Under MM-OCT, these agents have been detected above various levels of background tissue scattering demonstrating that these agents can provide added contrast to OCT through the magnetic signal. These agents were incubated with various cell lines with differing levels of alpha(v)beta(3) integrin receptor expression that were quantified using western blotting and fluorescent antibody immunohistochemical staining. The normal control cell line used was the CRL-4010. The breast cancer cell lines studied included CRL-2314, SK-BR-3, MCF-7, and 13762 MAT B III cells. These studies address the binding specificity and sensitivity of the RGD functionalized protein microspheres to the alpha(v)beta(3) integrin receptors. In addition, a quantitative analysis is being performed to correlate the relative levels of bound microspheres to the cells, measured through MM-OCT measurements and through their fluorescence signals of the microspheres, and the cell’s alpha(v)beta(3) integrin receptor expression derived from the western blot experiments. Preliminary results indicate that these agents have a higher affinity to the cancer cells over the normal epithelial cells and are also internalized by the cells and could have to potential to become localized targeted drug delivery vehicles. In an NMU carcinogen induced rat animal model, the targeted protein microspheres were injected in-vivo. These preliminary results, using a multi-spectral dark box imaging system, demonstrate the localization of the microspheres to the vasculature surrounding the tumor. These microspheres are being presented as a novel contrast agent to a novel high resolution imaging modality targeted at cancer.


Optical coherence tomography: the intraoperative assessment of lymph nodes in breast cancer

During breast-conserving surgeries, axillary lymph nodes draining from the primary tumor site are removed for disease staging. Although a high number of lymph nodes are often resected during sentinel and lymph-node dissections, only a relatively small percentage of nodes are found to be metastatic, a fact that must be weighed against potential complications such as lymphedema. Without a real-time in vivo or in situ intraoperative imaging tool to provide a microscopic assessment of the nodes, postoperative paraffin section histopathological analysis currently remains the gold standard in assessing the status of lymph nodes. This paper investigates the use of optical coherence tomography (OCT), a high-resolution real-time microscopic optical-imaging technique, for the intraoperative ex vivo imaging and assessment of axillary lymph nodes. Normal (13), reactive (1), and metastatic (3) lymph nodes from 17 human patients with breast cancer were imaged intraoperatively with OCT. These preliminary clinical studies have identified scattering changes in the cortex, relative to the capsule, which can be used to differentiate normal from reactive and metastatic nodes. These optical scattering changes are correlated with inflammatory and immunological changes observed in the follicles and germinal centers. These results suggest that intraoperative OCT has the potential to assess the real-time node status in situ, without having to physically resect and histologically process specimens to visualize microscopic features.


Intraoperative evaluation of breast tumor margins with optical coherence tomography

As breast cancer screening rates increase, smaller and more numerous lesions are being identified earlier, leading to more breast-conserving surgical procedures. Achieving a clean surgical margin represents a technical challenge with important clinical implications. Optical coherence tomography (OCT) is introduced as an intraoperative high-resolution imaging technique that assesses surgical breast tumor margins by providing real-time microscopic images up to 2 mm beneath the tissue surface. In a study of 37 patients split between training and study groups, OCT images covering 1 cm(2) regions were acquired from surgical margins of lumpectomy specimens, registered with ink, and correlated with corresponding histologic sections. A 17-patient training set used to establish standard imaging protocols and OCT evaluation criteria showed that areas of higher scattering tissue with a heterogeneous pattern were indicative of tumor cells and tumor tissue in contrast to lower scattering adipocytes found in normal breast tissue. The remaining 20 patients were enrolled into the feasibility study. Of these lumpectomy specimens, 11 were identified with a positive or close surgical margin and 9 were identified with a negative margin under OCT. Based on histologic findings, 9 true positives, 9 true negatives, 2 false positives, and 0 false negatives were found, yielding a sensitivity of 100% and specificity of 82%. These results show the potential of OCT as a real-time method for intraoperative margin assessment in breast-conserving surgeries.


Clinical feasibility of microscopically-guided breast needle biopsy using a fiber-optic probe with computer-aided detection

Needle biopsy of small or nonpalpable breast lesions has a high nondiagnostic sampling rate even when needle position is guided by stereotaxis or ultrasound. We assess the feasibility of using a near-infrared fiber optic probe and computer-aided detection for the microscopic guidance of needle breast biopsy procedures. Specimens from nine consented patients undergoing breast-conserving surgery were assessed intraoperatively using a needle device with an integrated fiber-optic probe capable of assessing two physical tissue properties highly correlated to pathology. Immediately following surgical resection, specimens were probed by inserting the optical biopsy needle device into the tissue, simulating the procedure used to position standard biopsy needles. Needle positions were marked and correlated with histology, which verified measurements obtained from 58 needle positions, including 40 in adipose and 18 in tumor tissue. This study yielded tissue classifications based on measurement of optical refractive index and scattering. Confidence-rating schemes yielded combined sensitivity of 89% (16/18) and specificity of 78% (31/40). Refractive index tests alone identified tumor tissue with a sensitivity of 83% (15/18) and specificity of 75% (30/40). Scattering profiles independently identified tumor tissue with a sensitivity of 61% (11/18) and specificity of 60% (24/40). These results show that a biopsy needle with an integrated fiber optic probe can be used to identify breast tumor tissue for sampling. Integration of this probe into current practices offers the potential to reduce nondiagnostic sampling rates by directly evaluating in situ microscopic tissue properties in real-time, before removal.