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Photodynamic quenched cathepsin activity based probes for cancer detection and macrophage targeted therapy.

Ben-Nun Y, Merquiol E, Brandis A, Turk B, Scherz A, Blum G - Theranostics (2015)

Bottom Line: Elevated cathepsins levels and activities are found in several types of human cancer, making them valuable biomarkers for detection and targeting therapeutics.Our qABPs carry a photosensitizer (PS), which is activated by light, resulting in oxidative stress and subsequent cell ablation, and a quencher that when removed by active cathepsins allow the PS to fluoresce and demonstrate PD properties.Our most powerful and stable PS-qABP, YBN14, consists of a selective cathepsin recognition sequence, a QC-1 quencher and a new bacteriochlorin derivative as a PS.

View Article: PubMed Central - PubMed

Affiliation: 1. The Institute of Drug Research, The School of Pharmacy, The Faculty of Medicine, Campus Ein Karem, The Hebrew University, Jerusalem, Israel.

ABSTRACT
Elevated cathepsins levels and activities are found in several types of human cancer, making them valuable biomarkers for detection and targeting therapeutics. We designed small molecule quenched activity-based probes (qABPs) that fluoresce upon activity-dependent covalent modification, yielding cell killing by Photodynamic Therapy (PDT). These novel molecules are highly selective theranostic probes that enable both detection and treatment of cancer with minimal side effects. Our qABPs carry a photosensitizer (PS), which is activated by light, resulting in oxidative stress and subsequent cell ablation, and a quencher that when removed by active cathepsins allow the PS to fluoresce and demonstrate PD properties. Our most powerful and stable PS-qABP, YBN14, consists of a selective cathepsin recognition sequence, a QC-1 quencher and a new bacteriochlorin derivative as a PS. YBN14 allowed rapid and selective non-invasive in vivo imaging of subcutaneous tumors and induced specific tumor macrophage apoptosis by light treatment, resulting in a substantial tumor shrinkage in an aggressive breast cancer mouse model. These results demonstrate for the first time that the PS-qABPs technology offers a functional theranostic tool, which can be applied to numerous tumor types and other inflammation-associated diseases.

No MeSH data available.


Related in: MedlinePlus

PDT in tumor-bearing mice. (a) 4T1 cells were injected subcutaneously in two separate locations on the back of BALB/c mice. After tumors were established (marked by arrows), YBN14 or bacteriochlorin (b-Chlo) were injected intravenous (via tail vein). Mice were imaged at indicated time as described in Figure 4. After acquiring the image at 8 and 16 hours the right tumor was treated with light (indicated with a thunder cartoon) while the left tumor was kept in the dark. (b) Mice were sacrificed 24 hours after injections and tumor, spleen, kidneys, and liver were extracted and imaged ex vivo using the same IVIS Kinetic equipped with a 710/760nm excitation/emission filter. Right panel shows average of organ fluorescence in (p/sec/cm2/sr)/(μW/cm2) units. (c) Parts of the tumors and organs were lysed and equal protein samples were spread by SDS-PAGE, followed by fluorescent scanning of the gel using an Odyssey Scanner 780/800nm. Specific in vivo cathepsins labeling by YBN14 is detected on gel. (d) Mice bearing a single tumor were treated with YBN14 or vehicle at indicated times. Light treatments were applied at 8 and 16 hrs post probe injection. Growth curve presenting the fold increase in tumor volume relative to volume measured at day 6, * p<0.05. (e) Part of tumors from (a) were frozen in OCT, sectioned, and stained with cleaved Caspaese-3 and F4/80 antibodies labeled with Cy5 and Cy3 respectively. Representative fluorescent scan acquired with an Olympus FV10i confocal microscope are presented, green- F4/80, Red- cleaved Caspase-3, blue- DAPI. Apoptosis indicated by cleaved caspase 3 was detected only in F4/80 macrophages of YBN14 and light treated tumors.
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Figure 5: PDT in tumor-bearing mice. (a) 4T1 cells were injected subcutaneously in two separate locations on the back of BALB/c mice. After tumors were established (marked by arrows), YBN14 or bacteriochlorin (b-Chlo) were injected intravenous (via tail vein). Mice were imaged at indicated time as described in Figure 4. After acquiring the image at 8 and 16 hours the right tumor was treated with light (indicated with a thunder cartoon) while the left tumor was kept in the dark. (b) Mice were sacrificed 24 hours after injections and tumor, spleen, kidneys, and liver were extracted and imaged ex vivo using the same IVIS Kinetic equipped with a 710/760nm excitation/emission filter. Right panel shows average of organ fluorescence in (p/sec/cm2/sr)/(μW/cm2) units. (c) Parts of the tumors and organs were lysed and equal protein samples were spread by SDS-PAGE, followed by fluorescent scanning of the gel using an Odyssey Scanner 780/800nm. Specific in vivo cathepsins labeling by YBN14 is detected on gel. (d) Mice bearing a single tumor were treated with YBN14 or vehicle at indicated times. Light treatments were applied at 8 and 16 hrs post probe injection. Growth curve presenting the fold increase in tumor volume relative to volume measured at day 6, * p<0.05. (e) Part of tumors from (a) were frozen in OCT, sectioned, and stained with cleaved Caspaese-3 and F4/80 antibodies labeled with Cy5 and Cy3 respectively. Representative fluorescent scan acquired with an Olympus FV10i confocal microscope are presented, green- F4/80, Red- cleaved Caspase-3, blue- DAPI. Apoptosis indicated by cleaved caspase 3 was detected only in F4/80 macrophages of YBN14 and light treated tumors.

Mentions: After establishing the optimal time point for light treatment, we evaluated the probe's ability to induce cell death after light treatment. We injected 4T1 tumor cells subcutaneously in two locations on the back of BALB/c mice. When tumors were established, YBN14 or bacteriochlorin were injected intravenously via the tail vein. Eight hours post probes injection a high fluorescent signal was detected in the tumors of YBN14 injected mice. At that time point one of the tumors was treated with light at 760 nm and the other tumor was covered, serving as a dark control. We repeated the light treatment of the same tumor at the 16 hour time point (Figure 5a). Mice were sacrificed 24 hours post probe injection, and then tumors and other major organs were excised and analyzed ex vivo. As suspected all organs of bacteriochlorin injected mice lacked detectable fluorescence. In contrast, mice injected with the quenched YBN14 probe showed significant fluorescence in organs with high cathepsin activity, i.e. kidney, liver and spleen. The tumors that were kept in dark showed significant fluorescence while the tumors that were light irradiated lacked fluorescence due to bleaching of the PS (Figure 5b). Tissue lysates were prepared and separated by SDS-PAGE, highly specific labeling of the cathepsins was detected by the fluorescent bands between 20-35 kDa, characteristic for these enzymes (Figure 5c).


Photodynamic quenched cathepsin activity based probes for cancer detection and macrophage targeted therapy.

Ben-Nun Y, Merquiol E, Brandis A, Turk B, Scherz A, Blum G - Theranostics (2015)

PDT in tumor-bearing mice. (a) 4T1 cells were injected subcutaneously in two separate locations on the back of BALB/c mice. After tumors were established (marked by arrows), YBN14 or bacteriochlorin (b-Chlo) were injected intravenous (via tail vein). Mice were imaged at indicated time as described in Figure 4. After acquiring the image at 8 and 16 hours the right tumor was treated with light (indicated with a thunder cartoon) while the left tumor was kept in the dark. (b) Mice were sacrificed 24 hours after injections and tumor, spleen, kidneys, and liver were extracted and imaged ex vivo using the same IVIS Kinetic equipped with a 710/760nm excitation/emission filter. Right panel shows average of organ fluorescence in (p/sec/cm2/sr)/(μW/cm2) units. (c) Parts of the tumors and organs were lysed and equal protein samples were spread by SDS-PAGE, followed by fluorescent scanning of the gel using an Odyssey Scanner 780/800nm. Specific in vivo cathepsins labeling by YBN14 is detected on gel. (d) Mice bearing a single tumor were treated with YBN14 or vehicle at indicated times. Light treatments were applied at 8 and 16 hrs post probe injection. Growth curve presenting the fold increase in tumor volume relative to volume measured at day 6, * p<0.05. (e) Part of tumors from (a) were frozen in OCT, sectioned, and stained with cleaved Caspaese-3 and F4/80 antibodies labeled with Cy5 and Cy3 respectively. Representative fluorescent scan acquired with an Olympus FV10i confocal microscope are presented, green- F4/80, Red- cleaved Caspase-3, blue- DAPI. Apoptosis indicated by cleaved caspase 3 was detected only in F4/80 macrophages of YBN14 and light treated tumors.
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Related In: Results  -  Collection

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Figure 5: PDT in tumor-bearing mice. (a) 4T1 cells were injected subcutaneously in two separate locations on the back of BALB/c mice. After tumors were established (marked by arrows), YBN14 or bacteriochlorin (b-Chlo) were injected intravenous (via tail vein). Mice were imaged at indicated time as described in Figure 4. After acquiring the image at 8 and 16 hours the right tumor was treated with light (indicated with a thunder cartoon) while the left tumor was kept in the dark. (b) Mice were sacrificed 24 hours after injections and tumor, spleen, kidneys, and liver were extracted and imaged ex vivo using the same IVIS Kinetic equipped with a 710/760nm excitation/emission filter. Right panel shows average of organ fluorescence in (p/sec/cm2/sr)/(μW/cm2) units. (c) Parts of the tumors and organs were lysed and equal protein samples were spread by SDS-PAGE, followed by fluorescent scanning of the gel using an Odyssey Scanner 780/800nm. Specific in vivo cathepsins labeling by YBN14 is detected on gel. (d) Mice bearing a single tumor were treated with YBN14 or vehicle at indicated times. Light treatments were applied at 8 and 16 hrs post probe injection. Growth curve presenting the fold increase in tumor volume relative to volume measured at day 6, * p<0.05. (e) Part of tumors from (a) were frozen in OCT, sectioned, and stained with cleaved Caspaese-3 and F4/80 antibodies labeled with Cy5 and Cy3 respectively. Representative fluorescent scan acquired with an Olympus FV10i confocal microscope are presented, green- F4/80, Red- cleaved Caspase-3, blue- DAPI. Apoptosis indicated by cleaved caspase 3 was detected only in F4/80 macrophages of YBN14 and light treated tumors.
Mentions: After establishing the optimal time point for light treatment, we evaluated the probe's ability to induce cell death after light treatment. We injected 4T1 tumor cells subcutaneously in two locations on the back of BALB/c mice. When tumors were established, YBN14 or bacteriochlorin were injected intravenously via the tail vein. Eight hours post probes injection a high fluorescent signal was detected in the tumors of YBN14 injected mice. At that time point one of the tumors was treated with light at 760 nm and the other tumor was covered, serving as a dark control. We repeated the light treatment of the same tumor at the 16 hour time point (Figure 5a). Mice were sacrificed 24 hours post probe injection, and then tumors and other major organs were excised and analyzed ex vivo. As suspected all organs of bacteriochlorin injected mice lacked detectable fluorescence. In contrast, mice injected with the quenched YBN14 probe showed significant fluorescence in organs with high cathepsin activity, i.e. kidney, liver and spleen. The tumors that were kept in dark showed significant fluorescence while the tumors that were light irradiated lacked fluorescence due to bleaching of the PS (Figure 5b). Tissue lysates were prepared and separated by SDS-PAGE, highly specific labeling of the cathepsins was detected by the fluorescent bands between 20-35 kDa, characteristic for these enzymes (Figure 5c).

Bottom Line: Elevated cathepsins levels and activities are found in several types of human cancer, making them valuable biomarkers for detection and targeting therapeutics.Our qABPs carry a photosensitizer (PS), which is activated by light, resulting in oxidative stress and subsequent cell ablation, and a quencher that when removed by active cathepsins allow the PS to fluoresce and demonstrate PD properties.Our most powerful and stable PS-qABP, YBN14, consists of a selective cathepsin recognition sequence, a QC-1 quencher and a new bacteriochlorin derivative as a PS.

View Article: PubMed Central - PubMed

Affiliation: 1. The Institute of Drug Research, The School of Pharmacy, The Faculty of Medicine, Campus Ein Karem, The Hebrew University, Jerusalem, Israel.

ABSTRACT
Elevated cathepsins levels and activities are found in several types of human cancer, making them valuable biomarkers for detection and targeting therapeutics. We designed small molecule quenched activity-based probes (qABPs) that fluoresce upon activity-dependent covalent modification, yielding cell killing by Photodynamic Therapy (PDT). These novel molecules are highly selective theranostic probes that enable both detection and treatment of cancer with minimal side effects. Our qABPs carry a photosensitizer (PS), which is activated by light, resulting in oxidative stress and subsequent cell ablation, and a quencher that when removed by active cathepsins allow the PS to fluoresce and demonstrate PD properties. Our most powerful and stable PS-qABP, YBN14, consists of a selective cathepsin recognition sequence, a QC-1 quencher and a new bacteriochlorin derivative as a PS. YBN14 allowed rapid and selective non-invasive in vivo imaging of subcutaneous tumors and induced specific tumor macrophage apoptosis by light treatment, resulting in a substantial tumor shrinkage in an aggressive breast cancer mouse model. These results demonstrate for the first time that the PS-qABPs technology offers a functional theranostic tool, which can be applied to numerous tumor types and other inflammation-associated diseases.

No MeSH data available.


Related in: MedlinePlus