Margarida Barroso

Fluorescence Lifetime Imaging for Quantification of Targeted Drug Delivery in Varying Tumor Microenvironments

Abstract Trastuzumab (TZM) is a monoclonal antibody that targets the human epidermal growth factor receptor 2 (HER2) and is clinically used for the treatment of HER2‐positive breast tumors. However, the tumor microenvironment can limit the access of TZM to the HER2 targets across the whole tumor and thereby compromising TZM's therapeutic efficacy. An imaging methodology that can non‐invasively quantify the binding of TZM‐HER2, which is required for therapeutic action, and distribution within tumors with varying tumor microenvironments is much needed. Near‐infrared (NIR) fluorescence lifetime (FLI) Forster Resonance Energy Transfer (FRET) is performed to measure TZM‐HER2 binding, using in vitro microscopy and in vivo widefield macroscopy, in HER2 overexpressing breast and ovarian cancer cells and tumor xenografts, respectively. Immunohistochemistry is used to validate in vivo imaging results. NIR FLI FRET in vitro microscopy data show variations in intracellular distribution of bound TZM in HER2‐positive breast AU565 and AU565 tumor‐passaged XTM cell lines in comparison to SKOV‐3 ovarian cancer cells. Macroscopy FLI (MFLI) FRET in vivo imaging data show that SKOV‐3 tumors display reduced TZM binding compared to AU565 and XTM tumors, as validated by ex vivo immunohistochemistry. Moreover, AU565/XTM and SKOV‐3 tumor xenografts display different amounts and distributions of TME components, such as collagen and vascularity. Therefore, these results suggest that SKOV‐3 tumors are refractory to TZM delivery due to their disrupted vasculature and increased collagen content. The study demonstrates that FLI is a powerful analytical tool to monitor the delivery of antibodydrugs both in cell cultures and in vivo live systems. Especially, MFLI FRET is a unique imaging modality that can directly quantify target engagement with the potential to elucidate the role of the TME in drug delivery efficacy in intact live tumor xenografts.

Contrast-enhanced photon-counting micro-CT of tumor xenograft models

Abstract Objective . Photon-counting micro-computed tomography (micro-CT) is a major advance in small animal preclinical imaging. Small molecule- and nanoparticle-based contrast agents have been widely used to enable the differentiation of liver tumors from surrounding tissues using photon-counting micro-CT. However, there is a notable gap in the application of these market-available agents to the imaging of breast and ovarian tumors using photon-counting micro-CT. Herein, we have used photon-counting micro-CT to determine the effectiveness of these contrast agents in differentiating ovarian and breast tumor xenografts in live, intact mice. Approach . Nude mice carrying different types of breast and ovarian tumor xenografts (AU565, MDA-MB-231 and SKOV-3 human cancer cells) were injected with ISOVUE-370 (a small molecule-based agent) or Exitron Nano 12000 (a nanoparticle-based agent) and subjected to photon-counting micro-CT. To improve tumor visualization using photon-counting micro-CT, we developed a novel color visualization method, which changes color tones to highlight contrast media distribution, offering a robust alternative to traditional material decomposition methods with less computational demand. Main results . Our in vivo experiments confirm the effectiveness of this color visualization approach, showing distinct enhancement characteristics for each contrast agent. Qualitative and quantitative analyses suggest that Exitron Nano 12000 provides superior vasculature enhancement and better quantitative consistency across scans, while ISOVUE-370 delivers a more comprehensive tumor enhancement but with significant variance between scans due to its short blood half-time. Further, a paired t-test on mean and standard deviation values within tumor volumes showed significant differences between the AU565 and SKOV-3 tumor models with the nanoparticle-based contrast agent ( p -values < 0.02), attributable to their distinct vascularity, as confirmed by immunohistochemical analysis. Significance . These findings underscore the utility of photon-counting micro-CT in non-invasively assessing the morphology and anatomy of different tumor xenografts, which is crucial for tumor characterization and longitudinal monitoring of tumor progression and response to treatments.