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Image-Based Quantification of Benzoporphyrin Derivative Uptake, Localization, and Photobleaching in 3D Tumor Models, for Optimization of PDT Parameters.

Glidden MD, Celli JP, Massodi I, Rizvi I, Pogue BW, Hasan T - Theranostics (2012)

Bottom Line: While the systematic optimization of these treatment parameters can be complex, it also provides multiple avenues for enhancement of PDT efficacy under diverse treatment conditions, provided that a rational framework is established to quantify the impact of parameter selection upon treatment response.We use this approach to visualize and quantify the uptake, localization, and photobleaching of the PS benzoporphyrin derivative monoacid ring-A (BPD) in a range of treatment conditions with varying uptake times as well as continuous and fractionated light delivery regimens in 3D cultures of AsPC-1 and PANC-1 cells.Quantification of the spatial profile of cell killing within multicellular nodules revealed that these conditions also achieve the highest depth of cytotoxicity along the radial axis of 3D nodules.

View Article: PubMed Central - PubMed

Affiliation: 1. Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA 02114, USA; ; 2. Department of Physics, University of Massachusetts Boston, Boston, MA 02125, USA;

ABSTRACT
Photodynamic therapy (PDT) is a light-based treatment modality in which wavelength specific activation of a photosensitizer (PS) generates cytotoxic response in the irradiated region. PDT response is critically dependent on several parameters including light dose, PS dose, uptake time, fluence rate, and the mode of light delivery. While the systematic optimization of these treatment parameters can be complex, it also provides multiple avenues for enhancement of PDT efficacy under diverse treatment conditions, provided that a rational framework is established to quantify the impact of parameter selection upon treatment response. Here we present a theranostic technique, combining the inherent ability of the PS to serve simultaneously as a therapeutic and imaging agent, with the use of image-based treatment assessment in three dimensional (3D) in vitro tumor models, to comprise a platform to evaluate the impact of PDT parameters on treatment outcomes. We use this approach to visualize and quantify the uptake, localization, and photobleaching of the PS benzoporphyrin derivative monoacid ring-A (BPD) in a range of treatment conditions with varying uptake times as well as continuous and fractionated light delivery regimens in 3D cultures of AsPC-1 and PANC-1 cells. Informed by photobleaching patterns and correlation with cytotoxic response, asymmetric fractionated light delivery at 4 hours BPD uptake was found to be the most effective regimen assessed. Quantification of the spatial profile of cell killing within multicellular nodules revealed that these conditions also achieve the highest depth of cytotoxicity along the radial axis of 3D nodules. The framework introduced here provides a means for systematic assessment of PDT treatment parameters in biologically relevant 3D tumor models with potential for broader application to other systems.

No MeSH data available.


Related in: MedlinePlus

BPD Uptake Curves for PANC-1 and AsPC-1 3D Cultures. Method by which photosensitizer uptake curves are obtained in the 3D model, beginning with (A) BPD fluorescence images of day 10 PANC-1 cultures taken 5 minutes after media change to BPD-free media (images from AsPC-1 cultures not shown for the sake of clarity). Quantified uptake curves were generated by taking the mean pixel intensities of each image and are presented for (B) day 12 AsPC-1 and (C) day 10 PANC-1 nodules. Ratiometric increases in (B) and (C) are reported as the ratio of the mean pixel intensities of images collected at 24 hours of uptake to those collected at 1 hour of uptake. All scale bars are 350 μm.
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Figure 2: BPD Uptake Curves for PANC-1 and AsPC-1 3D Cultures. Method by which photosensitizer uptake curves are obtained in the 3D model, beginning with (A) BPD fluorescence images of day 10 PANC-1 cultures taken 5 minutes after media change to BPD-free media (images from AsPC-1 cultures not shown for the sake of clarity). Quantified uptake curves were generated by taking the mean pixel intensities of each image and are presented for (B) day 12 AsPC-1 and (C) day 10 PANC-1 nodules. Ratiometric increases in (B) and (C) are reported as the ratio of the mean pixel intensities of images collected at 24 hours of uptake to those collected at 1 hour of uptake. All scale bars are 350 μm.

Mentions: These raw fluorescence images and the calculated photobleaching map thus provide rich and thorough information about PDT parameters. Firstly, PS fluorescence images collected pre-treatment provide a means to quantify not only mean, but nodule-by-nodule photosensitizer uptake and localization. Pixel intensity line scans (shown as white arrows in each enlarged nodule images) of single nodules reveal the photosensitizer's localization within the acini. It can be seen in Figure 1 that photosensitizer is localized primarily on the outer edges of the nodule (blue plot, left). From this information, uptake curves are obtained by imaging selected groups of nodules that are administered photosensitizer at different uptake times and then taking the mean pixel intensity of each image (see Figure 2). Similar information is obtained regarding the degree and localization of nodule-by-nodule photobleaching. The plot profile of the same nodule in the photobleaching map shows that not only is photosensitizer primarily located on the periphery of the nodule, but that photobleaching is highest in the same region (red plot, middle). In the live and dead fluorescence images under the “Imaging-based Viability Assessment” panel in Figure 1, it can be seen that the same nodule has nearly no viable cells in the analogous region where photobleaching was maximal. Consequently, the most viable cells survive in the core of the nodule where photobleaching was minimal.


Image-Based Quantification of Benzoporphyrin Derivative Uptake, Localization, and Photobleaching in 3D Tumor Models, for Optimization of PDT Parameters.

Glidden MD, Celli JP, Massodi I, Rizvi I, Pogue BW, Hasan T - Theranostics (2012)

BPD Uptake Curves for PANC-1 and AsPC-1 3D Cultures. Method by which photosensitizer uptake curves are obtained in the 3D model, beginning with (A) BPD fluorescence images of day 10 PANC-1 cultures taken 5 minutes after media change to BPD-free media (images from AsPC-1 cultures not shown for the sake of clarity). Quantified uptake curves were generated by taking the mean pixel intensities of each image and are presented for (B) day 12 AsPC-1 and (C) day 10 PANC-1 nodules. Ratiometric increases in (B) and (C) are reported as the ratio of the mean pixel intensities of images collected at 24 hours of uptake to those collected at 1 hour of uptake. All scale bars are 350 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475211&req=5

Figure 2: BPD Uptake Curves for PANC-1 and AsPC-1 3D Cultures. Method by which photosensitizer uptake curves are obtained in the 3D model, beginning with (A) BPD fluorescence images of day 10 PANC-1 cultures taken 5 minutes after media change to BPD-free media (images from AsPC-1 cultures not shown for the sake of clarity). Quantified uptake curves were generated by taking the mean pixel intensities of each image and are presented for (B) day 12 AsPC-1 and (C) day 10 PANC-1 nodules. Ratiometric increases in (B) and (C) are reported as the ratio of the mean pixel intensities of images collected at 24 hours of uptake to those collected at 1 hour of uptake. All scale bars are 350 μm.
Mentions: These raw fluorescence images and the calculated photobleaching map thus provide rich and thorough information about PDT parameters. Firstly, PS fluorescence images collected pre-treatment provide a means to quantify not only mean, but nodule-by-nodule photosensitizer uptake and localization. Pixel intensity line scans (shown as white arrows in each enlarged nodule images) of single nodules reveal the photosensitizer's localization within the acini. It can be seen in Figure 1 that photosensitizer is localized primarily on the outer edges of the nodule (blue plot, left). From this information, uptake curves are obtained by imaging selected groups of nodules that are administered photosensitizer at different uptake times and then taking the mean pixel intensity of each image (see Figure 2). Similar information is obtained regarding the degree and localization of nodule-by-nodule photobleaching. The plot profile of the same nodule in the photobleaching map shows that not only is photosensitizer primarily located on the periphery of the nodule, but that photobleaching is highest in the same region (red plot, middle). In the live and dead fluorescence images under the “Imaging-based Viability Assessment” panel in Figure 1, it can be seen that the same nodule has nearly no viable cells in the analogous region where photobleaching was maximal. Consequently, the most viable cells survive in the core of the nodule where photobleaching was minimal.

Bottom Line: While the systematic optimization of these treatment parameters can be complex, it also provides multiple avenues for enhancement of PDT efficacy under diverse treatment conditions, provided that a rational framework is established to quantify the impact of parameter selection upon treatment response.We use this approach to visualize and quantify the uptake, localization, and photobleaching of the PS benzoporphyrin derivative monoacid ring-A (BPD) in a range of treatment conditions with varying uptake times as well as continuous and fractionated light delivery regimens in 3D cultures of AsPC-1 and PANC-1 cells.Quantification of the spatial profile of cell killing within multicellular nodules revealed that these conditions also achieve the highest depth of cytotoxicity along the radial axis of 3D nodules.

View Article: PubMed Central - PubMed

Affiliation: 1. Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA 02114, USA; ; 2. Department of Physics, University of Massachusetts Boston, Boston, MA 02125, USA;

ABSTRACT
Photodynamic therapy (PDT) is a light-based treatment modality in which wavelength specific activation of a photosensitizer (PS) generates cytotoxic response in the irradiated region. PDT response is critically dependent on several parameters including light dose, PS dose, uptake time, fluence rate, and the mode of light delivery. While the systematic optimization of these treatment parameters can be complex, it also provides multiple avenues for enhancement of PDT efficacy under diverse treatment conditions, provided that a rational framework is established to quantify the impact of parameter selection upon treatment response. Here we present a theranostic technique, combining the inherent ability of the PS to serve simultaneously as a therapeutic and imaging agent, with the use of image-based treatment assessment in three dimensional (3D) in vitro tumor models, to comprise a platform to evaluate the impact of PDT parameters on treatment outcomes. We use this approach to visualize and quantify the uptake, localization, and photobleaching of the PS benzoporphyrin derivative monoacid ring-A (BPD) in a range of treatment conditions with varying uptake times as well as continuous and fractionated light delivery regimens in 3D cultures of AsPC-1 and PANC-1 cells. Informed by photobleaching patterns and correlation with cytotoxic response, asymmetric fractionated light delivery at 4 hours BPD uptake was found to be the most effective regimen assessed. Quantification of the spatial profile of cell killing within multicellular nodules revealed that these conditions also achieve the highest depth of cytotoxicity along the radial axis of 3D nodules. The framework introduced here provides a means for systematic assessment of PDT treatment parameters in biologically relevant 3D tumor models with potential for broader application to other systems.

No MeSH data available.


Related in: MedlinePlus