Info
Background:
Assessment of cell surface receptor expression is essential for guiding targeted therapies, including antibody-drug conjugates (ADCs), yet current approaches rely on invasive biopsies that provide limited information on temporal changes and spatial heterogeneity within tumors. Photoacoustic (PA) imaging addresses these challenges by providing point-of-care, noninvasive molecular and functional imaging at clinically relevant depths, including simultaneous assessment of oxygen saturation (SO2). Previously, PA imaging was used to assess intratumoral accumulation of anti-Folate Receptor alpha (aFRα)-targeted liposome-encapsulated J-aggregated indocyanine green (JICG) formulation (aFRα-PAtrace), in a murine ovarian cancer model [1]. Here, we evaluate a next-generation contrast agent comprising aFRα-targeted polymersome-encapsulated JICG (aFRα-PS), designed to prolong circulation time and enhance tumor accumulation for longitudinal molecular imaging in vivo [2]. JICG exhibits a strong absorbance peak at 890 nm, enabling simultaneous PA imaging of targeted agent accumulation and SO2.
Hypothesis/Objective:
This study evaluated the correlation between PA signal from aFRα-PS and tumor FRα expression measured by quantitative flow cytometry (FC), supporting the potential of PA imaging as a noninvasive surrogate for receptor quantification.
Methods:
An orthotopic ovarian cancer model was established by inoculating athymic nude mice (n=9) with SKOV3IP-Luc cells. When tumors reached ~ 5mm in diameter, mice received an intravenous injection of 100 µL of aFRα-PS (optical density range: 86-137 O.D.). Approximately one-hour post-injection, whole-animal volumetric PA imaging was performed using the MSOT inVision 256-TF system (iThera Medical). Image post-processing included manual segmentation of tumor regions of interest (ROIs) and optimization of spectral unmixing by evaluating multiple wavelength combinations and parameters, including post- versus pre-injection signal difference, to maximize specificity of PS signal extraction; PA signal was normalized by the injected O.D. value prior to correlation analysis. Following imaging, tumors were harvested, dissociated into single-cell suspensions, and analyzed by FC to quantify FRα surface expression. A standardized gating strategy was applied, incorporating sequential exclusion of debris, doublets, and non-viable cells to ensure objective and reproducible identification of tumor cell populations. FRα expression was quantified as antibody binding capacity (ABC) per tumor cell using a calibration bead-based approach, and mean ABC values were compared across all study animals.
Results:
PA imaging of aFRα-PS revealed signal enhancement within tumor ROIs approximately one-hour post-injection, with spatially distinct accumulation patterns observed across individual mice. FRα expression varied substantially across tumor dissociates, consistent with heterogeneous receptor expression among tumors. Across all tumors (n=9), PA signal significantly and positively correlated with ex vivo FRα expression measured by FC (Pearson correlation; one-tailed test; p = 0.027), demonstrating that molecular PA imaging reflects tumor receptor density in vivo. At higher levels of receptor expression and corresponding nanoparticle accumulation, near-field signal attenuation was observed, suggesting that the spatial extent of ROI analysis may require adjustment as a function of tumor uptake and labeling density to ensure accurate signal quantification.
Conclusion:
These findings demonstrate that molecular PA imaging using aFRα-PS enables noninvasive assessment of FRα expression in an orthotopic ovarian cancer model. Across all tumors, which presented heterogeneous receptor expression, in vivo PA signal showed a significant positive correlation with ex vivo FC–based receptor quantification, supporting PA imaging as a surrogate measure of tumor receptor density. This approach establishes a foundation for noninvasive, longitudinal molecular stratification of receptor-driven malignancies.
Image/Figure:
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Image/Figure Caption:
Molecular PA imaging of aFRα-PS correlates with ex vivo flow cytometry (FC)–based FRα expression in an orthotopic ovarian cancer model.
(A) Spectrally unmixed PS signal (green) overlaid on anatomical photoacoustic (PA) images revealed signal enhancement within tumor regions of interest (ROIs) approximately one-hour post-injection, with heterogeneous intratumoral accumulation observed across individual mice.
(B) Flow cytometry (FC)-based quantification of FRα surface expression from dissociated tumors. A standardized gating strategy was applied to objectively identify viable tumor cell populations. FRα expression was quantified as antibody binding capacity (ABC) per cell using calibration beads. Mean ABC values per tumor (n=9) demonstrated inter-tumor variability in FRα expression.
(C) Correlation between in vivo aFRα-PS PA signal and ex vivo FRα expression quantified by FC across orthotopic ovarian tumors. A significant positive linear correlation was observed (Pearson correlation; one-tailed test; p = 0.027), indicating that molecular PA signal quantitatively reflects tumor-level FRα expression and supports PA imaging as a noninvasive surrogate for receptor quantification in vivo.
Author
Paula Aguilera Rodriguez
The University of Texas MD Anderson Cancer Center