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. 2013 Oct;1830(10):4594-603.
doi: 10.1016/j.bbagen.2013.05.043. Epub 2013 Jun 7.

Vascular targeting to the SST2 receptor improves the therapeutic response to near-IR two-photon activated PDT for deep-tissue cancer treatment

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Vascular targeting to the SST2 receptor improves the therapeutic response to near-IR two-photon activated PDT for deep-tissue cancer treatment

Jean R Starkey et al. Biochim Biophys Acta. 2013 Oct.

Abstract

Background: Broader clinical acceptance of photodynamic therapy is currently hindered by (a) poor depth efficacy, and (b) predisposition towards establishment of an angiogenic environment during the treatment. Improved depth efficacy is being sought by exploiting the NIR tissue transparency window and by photo-activation using two-photon absorption (2PA). Here, we use two-photon activation of PDT sensitizers, untargeted and targeted to SST2 receptors or EGF receptors, to achieve deep tissue treatment.

Methods: Human tumor lines, positive or negative for SST2r expression were used, as well as murine 3LL cells and bovine aortic endothelial cells. Expression of SST2 receptors on cancer cells and tumor vasculature was evaluated in vitro and frozen xenograft sections. PDT effects on tumor blood flow were followed using in vivo scanning after intravenous injection of FITC conjugated dextran 150K. Dependence of the PDT efficacy on the laser pulse duration was evaluated. Effectiveness of targeting to vascular SST2 receptors was compared to that of EGF receptors, or no targeting.

Results: Tumor vasculature stained for SST2 receptors even in tumors from SST2 receptor negative cell lines, and SST2r targeted PDT led to tumor vascular shutdown. Stretching the pulse from ~120fs to ~3ps led to loss of the PDT efficacy especially at greater depth. PDT targeted to SST2 receptors was much more effective than untargeted PDT or PDT targeted to EGF receptors.

General significance: The use of octreotate to target SST2 receptors expressed on tumor vessels is an excellent approach to PDT with few recurrences and some long term cures.

Keywords: 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; ATCC; American type culture collection; BAE; CMF; DMEM; Dulbecco's minimal essential medium; EDC; EDTA; EGF; Ethylenediaminetetraacetic acid; FITC; GE11 peptide; Laser pulse; NIR; PBS; PDT; Photodynamic therapy; SCID; SST2r; Somatostatin receptor 2; UCNP; VEGF; Vascular shutdown; YHWYGYTPQNVI; bovine aortic endothelial; calcium and magnesium free saline; epidermal growth factor; fluorescein isothiocyanate; near infrared; phosphate buffered saline; photodynamic therapy; severe combined immunodeficient; somatostatin type 2 receptor; upconversion nanoparticle; vascular endothelial growth factor.

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Conflict of interest statement

The authors report the following potential conflicts of interest: A.K. Rebane and J.R. Starkey have 8.49% and 4.72% ownership interests respectively in SensoPath Technologies Inc.

Figures

Fig. 1
Fig. 1
SCID mouse receiving NIR laser activation. The watch glass on the side of the mouse along with added drops of glycerin reduced scattering on the optical interface between the laser beam and the mouse, and also aided heat dissipation at the skin surface.
Fig. 2
Fig. 2
Comparison of the regression of tumors using our normal laser parameters for deep PDT treatment with pulse lengthened (10 fold) laser parameters. Treated through the mouse = formula image treated directly = formula image Panel A = FaDu xenograft subcutaneous tumors induced as described in materials and methods, treated using our normal femtosecond laser pulse protocol for PDT. Panel B = FaDu xenograft subcutaneous tumors induced as described in materials and methods, treated using 10 fold pulse elongation of the laser beam. Panel C = 3LL subcutaneous tumors induced as described in materials and methods, treated using 10 fold pulse elongation of the laser beam. Note that the last day possible for tumor measurement for the group treated through the mouse was day +8 after laser treatment. This was due to aggressive growth of the tumor in this group.
Fig. 3
Fig. 3
SST2 receptor expression in vitro. T47D human breast carcinoma cells (panels A, B, and C ) and bovine aortic endothelial cells (D) were propagated on glass coverslips as described in materials and methods. Panels A) and B) were stained for SST2 receptors as described in materials and methods. Panel A) was fixed with acetone then stained with the primary antibody while panel B) was first stained with the primary antibody and then fixed with acetone. Panel C) was stained with the primary antibody in the presence of competing excess octreotate peptide. Panel D) was stained with biotinylated octreotate, then developed with neutravidin secondary reagents as described in materials and methods. Bar = 20 µm. N = nucleus.
Fig. 4
Fig. 4
Staining of blood vessels in tumor tissues. Panels A) and B) are samples from human SST2 receptor positive NCI H69 human xenograft tissue in SCID mice. Panel C) is from the SST2 receptor negative xenograft A549 human lung tumor in SCID mice. Panel A) is stained for factor 8 associated antigen, while panels B) and C) are stained for SST2 receptors as described in materials and methods. Bar = 750 µm.
Fig. 5
Fig. 5
In vivo imaging scans of tumor blood flow in NCI H69 xenograft tumors in SCID mice. 150K molecular weight FITC dextran was used to monitor tumor blood flow as described in materials and methods. NCI H69 xenograft subcutaneous tumors were used. Panel A) shows the extravasation of the dextran tracer from leaky vessels in the tumor tissue 40 minutes after intravenous tracer injection via the tail vein. Panel B) shows the effect on blood flow 6 hours after PDT treatment. Panel C) shows the blood flow pattern 48 hours before PDT treatment. Panel D) shows the blood flow pattern 6 hours after PDT treatment when the sensitizer was injected under conditions of octreotate blockade as described in materials and methods. Panel E) shows a picture of the tumor bearing mouse imaged in panels A) and B). The red circle outlines the tumor. Panel F) shows the pattern of fluorescence seen in a control tumor at increasing times after intravenous injection of FITC conjugated dextran 150K. Bar = 1cm. Red star = area of FITC fluorescence.
Fig. 6
Fig. 6
Comparison of the effectiveness of PDT treatments using different targeting peptides or untargeted PDT. All groups of mice carried subcutaneous FADU xenografts in the flank region. Neither the FaDu cell line nor the xenografts induced by this line in SCID mice were shown to express the SST2 receptor as described in materials and methods. Panel A) tumors received treatment with untargeted sensitizer = formula image, tumors received PDT using sensitizer targeted to the EGF receptor = formula image, tumors received PDT using sensitizer targeted to the SST2 receptor = formula image tumors received PDT using a 50:50 mixture of sensitizer targeted to the EGF receptor and sensitizer targeted to the SST2 receptor = formula image. Panel B) tumors received no treatment (control group for groups treated with targeted sensitizers in panel A) = formula image, tumors received no treatment (control group for the group treated using untargeted sensitizer in panel A) = formula image.

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