Using a dual MRI-FMT imaging approach, we confirmed the feasibility and sensitivity of detection of tumor-associated biologic activities in ovarian carcinomas in living mice by FMT imaging with fluorescent molecular imaging probes. For this study, we chose probes based on their relevance to EOC. Among these, the ProSense, MMPSense, and IntegriSense probes were considered to be highly relevant based on prior studies showing high levels of expression and activity of cathepsins, MMPs, and integrin αv
in human EOCs [20–27,30–35
]. Although immunohistochemical staining is commonly used for detection of proteases in tumor tissues, the mere presence of these proteins does not provide information about protease activity. An advantage of activatable fluorescent probes is that the probes are fluorescently quenched until they are cleaved by active proteases. Thus, probe activation and retention in tumors are directly reflective of tumor-associated protease activity. The fluorescent molecular probes used detected cathepsin and MMP activity and integrin αv
binding in ovarian tumors but not in normal wild-type ovaries. Quantitative assessments showed that cathepsin activity and integrin αv
binding were strongly correlated with tumor volume. MMP activity was readily detected in ovarian tumors; however, the correlation with tumor volume was not as strong. This may be related to differences in: 1) overall MMP activity in tumors, 2) lack of uniform MMP activity in tumors, or 3) intrinsic differences in the imaging probes properties (e.g., inherent fluorescent signal strength and/or capacity for tumor localization and penetration).
Advances in optical molecular imaging technologies offer the possibility of improved or alternative strategies for disease detection and staging and for the identification and observation of molecules and/or biologic activities associated with tumor initiation, progression and response to therapy. Importantly, in the current study, fluorescent probes targeting tumor-associated protease activity allowed in vivo detection of microscopic tumors in mice before enlargement of the ovary detectable by MRI. In addition, FMT coupled with a fluorescent probe detecting integrin αvβ3 binding enabled in vivo detection of both tumor progression and response to chemotherapy. The αvβ3 integrin binding signal detected in ovarian tumors may be from tumor cells, tumor vasculature or both. However, the FMT2500 instrumentation is not sufficiently sensitive to discriminate between these possibilities.
The findings in this study support additional investigation of molecular imaging for in vivo
early detection of ovarian tumors and for detection of functional response to targeted therapy. Some targeted and immune-based therapeutic agents show efficacy in the absence of radiologic evidence of tumor regression or in some cases after initial evidence of progressive disease [36,37
]. Therefore, optical assessments of biologic activities within tumors may provide relevant information regarding response to therapy in the absence of detectable changes in tumor volume achieved by anatomic imaging. Although beyond the scope of the present study, demonstration of value added by FMT-based imaging to the already highly sensitive and reliable anatomic imaging provided by MRI will require additional studies, the success of which will require the identification of a molecular targeted agent with significant antitumor activity for EOC and the availability of fluorescent probes that bind to or are activated by relevant downstream biologic mediators of the pathway targeted.
Until recently, in vivo
optical imaging strategies primarily relied on planar imaging methods (e.g., fluorescence reflectance or bioluminescent imaging) that, although informative, are nonquantitative and subject to significant limitations of light penetration and scattering in tissues [13
]. Improvements in tomographic optical imaging technology and fluorescent imaging probes with high target-to-background ratios and low tissue autofluorescence led to the availability of FMT instrumentation and its application for small animal imaging [38
]. Whereas FMT instrumentation theoretically enables imaging of fluorescent signals at depths more than 500 µm, there is little available information regarding in vivo
fluorescent molecular imaging of ovarian tumors in mice. Previous studies in mice with xenografts of human EOC cells demonstrated the feasibility of optical imaging using fluorescent probes for detection of tumor nodules but largely relied on ex vivo
]. The results of this study demonstrate for the first time the feasibility, sensitivity, and accuracy of fluorescent imaging probes and FMT for in vivo
detection of spontaneous ovarian tumors in black mice and for quantification of tumor-associated biologic activities.
Because FMT was a new approach for in vivo
imaging of spontaneous ovarian tumors, the combined imaging strategy increased the accuracy of detection and quantification of tumor-specific fluorescent probe activation/retention. The major challenge in image alignment was due to the gravitational effects on the organs in mice imaged in the vertical position for MRI compared with the horizontal position for FMT. Co-administration of AnnexinVivo to mark the kidneys for image alignment helped overcome this challenge. The observation of high AnnexinVivo probe accumulation in the kidneys is consistent with previous reports showing increased apoptosis and accumulation of exposed phosphatidyl serine in the proximal tubules of kidney after isoflurane inhalation [41
] as well as studies using technetium 99m-conjugated annexin V imaging agents showing that these agents are retained in the kidneys of mice and humans [42,43
]. Given the proximal location of kidneys to the ovaries, this approach was more effective than external installation of fiducial markers and allowed the use of the same anatomic structure as a fiducial for serial images. The imaging methods described in this work will be easily adaptable to imaging mice with orthotopic implants of human ovarian cancer cells or patient tumor tissue in the intrabursal space [17
Technical limitations related to the depth of tissue imaging make clinical adaptation of FMT imaging unlikely. However, the development of combination fluorescent and white light endoscopes has allowed in vivo
imaging of disseminated peritoneal tumor nodules using cathepsin activatable or small-molecule fluorescent imaging probes [44,45
]. The use of fluorescent imaging probes with high tumor-to-background ratios results in increased sensitivity and specificity in the detection of tumor nodules compared to white light imaging. Continued advancements in clinical adaptation of endoscopic fluorescent imaging technology and approval of activatable and/or targeted fluorescent probes for clinical use are predicted to improve surgical staging and optimal surgical debulking by detection of small disseminated lesions that are not visible by conventional white light imaging.
The need for alternative therapies for EOC is underscored by the common development of resistance to standard cytotoxic chemotherapy and the persistently high mortality rate associated with advanced stage ovarian cancer [46
]. Many targeted molecular therapies are available, several of which have been evaluated in clinical trials; however, there are few examples with demonstrated clinical efficacy for the majority of patients. Given the high degree of tumor heterogeneity [4
], EOC treatment may require a more individualized approach. High-throughput genomic and proteomic analyses of individual tumor specimens may identify genes and signaling pathways that contribute to cancer progression, but determining the most effective single agents or combinations remains a significant challenge. Studies in mouse models have the potential to facilitate the rapid evaluation of novel agents and effective drug combinations. Whereas the standard assessment of response to therapy is tumor regression, disease stabilization may be an acceptable clinical outcome. Multimodal imaging approaches for evaluation of changes in metabolic or biologic activity of the tumor may suggest a positive response to treatment. Further development of fluorescent molecular imaging probes is predicted to result in additional agents that can be used as biomarkers of functional response to a variety of targeted therapies. Importantly, the use of FMT for fluorescent imaging in preclinical models can provide quantitative information regarding specific probe activation and retention as well as response to therapy and is predicted to have translational applications for detection, staging, and treatment of ovarian cancer.