Fluorescence Lifetime Imaging for Quantification of Targeted Drug Delivery in Varying Tumor Microenvironments
Amit Verma1, Vikas Pandey2, Catherine Sherry1
1Department of Molecular and Cellular Physiology, Albany Medical College, Albany, NY, 12208, USA.
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.
Insights
Near-infrared fluorescence lifetime (FLI) FRET imaging quantifies Trastuzumab (TZM) binding to HER2-positive tumors. It reveals that tumor microenvironment factors like collagen and vasculature impact TZM delivery and efficacy, offering a new tool for antibody drug monitoring.
Area of Science:
- Biomedical Imaging
- Oncology
- Pharmacology
Background:
- Trastuzumab (TZM) is a monoclonal antibody targeting HER2 for HER2-positive breast cancer.
- Tumor microenvironment (TME) can impede TZM access to HER2 targets, limiting therapeutic efficacy.
- Non-invasive methods to quantify TZM-HER2 binding and distribution within tumors are needed.
Purpose of the Study:
- To develop and apply Near-Infrared (NIR) fluorescence lifetime (FLI) Förster Resonance Energy Transfer (FRET) imaging.
- To non-invasively quantify TZM-HER2 binding and distribution in HER2-overexpressing cancer models.
- To investigate the influence of the TME on TZM delivery and therapeutic efficacy.
Main Methods:
- In vitro microscopy and in vivo widefield macroscopy using NIR FLI FRET.
- Assessment in HER2-positive breast (AU565, XTM) and ovarian (SKOV-3) cancer cells and tumor xenografts.
- Immunohistochemistry for validation of in vivo imaging results and TME component analysis (collagen, vascularity).
Main Results:
- In vitro FLI FRET showed variations in intracellular TZM distribution in different cell lines.
- In vivo Macroscopy FLI (MFLI) FRET imaging revealed reduced TZM binding in SKOV-3 tumors compared to AU565/XTM tumors.
- SKOV-3 tumors exhibited disrupted vasculature and increased collagen, correlating with reduced TZM binding and suggesting refractoriness to TZM delivery.
Conclusions:
- FLI is a powerful tool for monitoring antibody-drug delivery in cell cultures and in vivo.
- MFLI FRET can directly quantify target engagement and elucidate the role of the TME in drug delivery efficacy.
- Tumor microenvironment characteristics significantly influence Trastuzumab delivery and efficacy in HER2-positive cancers.
