Denise K. Feyes

Phthalocyanine 4 (Pc 4) Photodynamic Therapy of Human OVCAR‐3 Tumor Xenografts

Abstract— Photodynamic therapy (PDT) is a cancer treatment modality utilizing a photosensitizer, light and oxygen. Photodynamic therapy with Photofrin® has been approved by the US. Food and Drug Administration for treatment of advanced esophageal and early lung cancer. Because of certain drawbacks associated with the use of Photofrin, there is a need to identify new photosensitizers for human use. The photosensitizer Pc 4 (HOSiPc‐OSi[CH3]2[CH2]3N[CH3]2) has yielded promising PDT effects in many in vitro and in vivo systems. The aim of this study was to assess the usefulness of Pc 4 as a PDT photosensitizer for a human tumor grown as a xenograft in athymic nude mice. The ovarian epithelial carcinoma (OVCAR‐3) was heterotransplanted subcutaneously in athymic nude mice. Sixty mice bearing OVCAR‐3 tumors (∼80–130 mm3) were divided into six groups of 10 animals each, three for controls and three for treatment. The Pc 4 was given by tail vein injection, and 48 h later a 1 cm area encompassing the tumor was irradiated with light from a diode laser coupled to a fiberoptic terminating in a microlens (Λ= 672 nm, 150 J/cm2,150 mW/cm2). Tumors of control animals receiving no treatment, light alone or Pc 4 alone continued to grow. Of animals receiving 0.4 mg/kg Pc 4 and light, one (10%) had a complete response and was cured (no regrowth up to 90 days post‐PDT), while all others (90%) had a partial response and were delayed in regrowth. Of animals receiving 0.6 mg/kg Pc 4 and light, eight (80%) had a complete response, and two of these were cured. Of animals receiving 1.0 mg/kg Pc 4 and light, six (60%) had a complete response, and two of these were cured. In additional experiments, tumors from animals treated with Pc 4 (1 mg/kg) and light were removed 15, 30, 60 and 180 min post‐PDT, and from these tumors DNA and protein were extracted. Agarose gel electrophoresis revealed the presence of apoptotic DNA fragmentation as early as 15 min post‐PDT. Western blotting showed the cleavage of the 116 kDa native poly (ADP‐ribose) polymerase (PARP) into fragments of ∼90 kDa, another indication of apoptosis, and the presence of p21/WAFl/CIPl (p21) in all PDT‐treated tumors. These changes did not occur in control tumors. Pc 4 appears to be an effective photosensitizer for PDT of human tumors grown as xenografts in nude mice. Early apoptosis, as revealed by PARP cleavage, DNA fragmentation and p21 overexpression, may be responsible for the excellent Pc 4‐PDT response. Clinical trials of Pc 4‐PDT are warranted.

Apoptosis is an early event during phthalocyanine photodynamic therapy-induced ablation of chemically induced squamous papillomas in mouse skin.

Abstract— Photodynamic therapy (PDT) is a promising new modality to treat malignant neoplasms including superficial skin cancers. In our search for an ideal photosensitizer for PDT, Pc 4, a silicon phthalocyanine, has shown promising results both in in vitro assays and in implanted tumors. In this study we assessed the efficacy of Pc 4 PDT in the ablation of murine skin tumors; and the evidence for apoptosis during tumor ablation was also obtained. The Pc 4 was administered through tail vein injection to SENCAR mice bearing chemically induced squamous papillomas, and 24 h later the lesions were illuminated with an argon ion‐pumped dye laser tuned at 675 nm for a total light dose of 135 J/cm2. Within 72‐96 h, almost complete tumor shrinkage occurred; no tumor regrowth was observed up to 90 days post‐PDT. As evident by nucleosome‐size DNA fragmentation, appearance of apoptotic bodies in hematoxylin and eosin staining and direct immunoperoxidase detection of digoxigenin‐labeled genomic DNA in sections, apoptosis was clearly evident 6 h post‐PDT at which time tumor shrinkage was less than 30%. The apoptotic bodies, as evident by the condensation of chromatin material around the periphery of the nucleus and increased vacuolization of the cytoplasm, were also observed in electron microscopic studies of the tumor tissues following Pc 4 PDT. The extent of apoptosis was greater at 15 h than at 6 and 10 h post‐PDT. Taken together, our results clearly show that Pc 4 may be an effective photosensitizer for PDT of nonmelanoma skin cancer, and that apoptosis is an early event during this process.

Deformable and rigid registration of MRI and microPET images for photodynamic therapy of cancer in mice

We are investigating imaging techniques to study the tumor response to photodynamic therapy (PDT). Positron emission tomography (PET) can provide physiological and functional information. High-resolution magnetic resonance imaging (MRI) can provide anatomical and morphological changes. Image registration can combine MRI and PET images for improved tumor monitoring. In this study, we acquired high-resolution MRI and microPET 18F-fluorodeoxyglucose (FDG) images from C3H mice with RIF-1 tumors that were treated with Pc 4-based PDT. We developed two registration methods for this application. For registration of the whole mouse body, we used an automatic three-dimensional, normalized mutual information algorithm. For tumor registration, we developed a finite element model (FEM)-based deformable registration scheme. To assess the quality of whole body registration, we performed slice-by-slice review of both image volumes; manually segmented feature organs, such as the left and right kidneys and the bladder, in each slice; and computed the distance between corresponding centroids. Over 40 volume registration experiments were performed with MRI and microPET images. The distance between corresponding centroids of organs was 1.5 +/- 0.4 mm which is about 2 pixels of microPET images. The mean volume overlap ratios for tumors were 94.7% and 86.3% for the deformable and rigid registration methods, respectively. Registration of high-resolution MRI and microPET images combines anatomical and functional information of the tumors and provides a useful tool for evaluating photodynamic therapy.

Apoptosis mechanisms related to the increased sensitivity of Jurkat T-cells vs A431 epidermoid cells to photodynamic therapy with the phthalocyanine Pc 4.

To examine the clinical applicability of Pc 4, a promising second‐generation photosensitizer, for the photodynamic treatment of lymphocyte‐mediated skin diseases, we studied the A431 and Jurkat cell lines, commonly used as surrogates for human keratinocyte‐derived carcinomas and lymphocytes, respectively. As revealed by ethyl acetate extraction and absorption spectrophotometry, uptake of Pc 4 into the two cell lines was linear with Pc 4 concentration and similar on a per cell basis but greater in Jurkat cells on a per mass basis. Flow cytometry showed that uptake was linear at low doses; variations in the dose–response for uptake measured by fluorescence supported differential aggregation of Pc 4 in the two cell types. As detected by confocal microscopy, Pc 4 localized to mitochondria and endoplasmic reticulum in both cell lines. Jurkat cells were much more sensitive to the lethal effects of phthalocyanine photodynamic therapy (Pc 4‐PDT) than were A431 cells, as measured by a tetrazolium dye reduction assay, and more readily underwent morphological apoptosis. In a search for molecular factors to explain the greater photosensitivity of Jurkat cells, the fate of important Bcl‐2 family members was monitored. Jurkat cells were more sensitive to the induction of immediate photodamage to Bcl‐2, but the difference was insufficient to account fully for their greater sensitivity. The antiapoptotic protein Mcl‐1 was extensively cleaved in a dose‐ and caspase‐dependent manner in Jurkat, but not in A431, cells exposed to Pc 4‐PDT. Thus, the greater killing by Pc 4‐PDT in Jurkat compared with A431 cells correlated with greater Bcl‐2 photodamage and more strongly to the more extensive Mcl‐1 degradation. Pc 4‐PDT may offer therapeutic advantages in targeting inflammatory cells over normal keratinocytes in the treatment of T‐cell‐mediated skin diseases, such as cutaneous lymphomas, dermatitis, lichenoid tissue reactions and psoriasis, and it will be instructive to evaluate the role of Bcl‐2 family proteins, especially Mcl‐1, in the therapeutic response.

Perspectives of Photodynamic Therapy for Skin Diseases

Photodynamic therapy (PDT) is largely an experimental modality for the treatment of neoplastic and selected nonneoplastic diseases. This therapeutic procedure, through a cascade of events, leads to cell killing. In the past few years, dermatology has taken advantage of PDT for the treatment of skin cancer and other skin diseases. The skin has considerable attributes over many other organs for the application of PDT. These include the accessibility to all three PDT essential requirements; the drug (photosensitizing agent), visible light and oxygen. The major benefit of experimental PDT in dermatology is the ability to assess the clinical response visually and the relative ease in obtaining biopsies for precise biochemical and histological analysis. Currently, PDT has received approval worldwide for the ablation of various tumor types. In the United States, the Food and Drug Administration has approved PDT for the treatment of advanced esophageal cancer and selected patients with lung cancer. Clinical trials, employing several types of photosensitizers for PDT, are ongoing for a variety of dermatological lesions. This review summarizes current knowledge of PDT in dermatology and highlights future perspectives of this modality for effective management of skin diseases.

In vivo small animal imaging for early assessment of therapeutic efficacy of photodynamic therapy for prostate cancer

We are developing in vivo small animal imaging techniques that can measure early effects of photodynamic therapy (PDT) for prostate cancer. PDT is an emerging therapeutic modality that continues to show promise in the treatment of cancer. At our institution, a new second-generation photosensitizing drug, the silicon phthalocyanine Pc 4, has been developed and evaluated at the Case Comprehensive Cancer Center. In this study, we are developing magnetic resonance imaging (MRI) techniques that provide therapy monitoring and early assessment of tumor response to PDT. We generated human prostate cancer xenografts in athymic nude mice. For the imaging experiments, we used a highfield 9.4-T small animal MR scanner (Bruker Biospec). High-resolution MR images were acquired from the treated and control tumors pre- and post-PDT and 24 hr after PDT. We utilized multi-slice multi-echo (MSME) MR sequences. During imaging acquisitions, the animals were anesthetized with a continuous supply of 2% isoflurane in oxygen and were continuously monitored for respiration and temperature. After imaging experiments, we manually segmented the tumors on each image slice for quantitative image analyses. We computed three-dimensional T2 maps for the tumor regions from the MSME images. We plotted the histograms of the T2 maps for each tumor pre- and post-PDT and 24 hr after PDT. After the imaging and PDT experiments, we dissected the tumor tissues and used the histologic slides to validate the MR images. In this study, six mice with human prostate cancer tumors were imaged and treated at the Case Center for Imaging Research. The T2 values of treated tumors increased by 24 ± 14% 24 hr after the therapy. The control tumors did not demonstrate significant changes of the T2 values. Inflammation and necrosis were observed within the treated tumors 24 hour after the treatment. Preliminary results show that Pc 4-PDT is effective for the treatment of human prostate cancer in mice. The small animal MR imaging provides a useful tool to evaluate early tumor response to photodynamic therapy in mice.

Optimal gadolinium dose level for magnetic resonance imaging (MRI) contrast enhancement of U87-derived tumors in athymic nude rats for the assessment of photodynamic therapy

This study aims to determine the effect of varying gadopentetate dimeglumine (Gd-DTPA) dose on Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI) tracking of brain tumor photodynamic therapy (PDT) outcome. Methods: We injected 2.5 x 105 U87 cells (derived from human malignant glioma) into the brains of six athymic nude rats. After 9, 12, and 13 days DCE-MRI images were acquired on a 9.4 T micro-MRI scanner before and after administration of 100, 150, or 200 μL of Gd-DTPA. Results: Tumor region normalized DCE-MRI scan enhancement at peak was: 1.217 over baseline (0.018 Standard Error [SE]) at the 100 μL dose, 1.339 (0.013 SE) at the 150 μL dose, and 1.287 (0.014 SE) at the 200 μL dose. DCE-MRI peak tumor enhancement at the 150 μL dose was significantly greater than both the 100 μL dose (p < 3.323E-08) and 200 μL dose (p < 0.0007396). Discussion: In this preliminary study, the 150 μL Gd-DTPA dose provided the greatest T1 weighted contrast enhancement, while minimizing negative T2* effects, in DCE-MRI scans of U87-derived tumors. Maximizing Gd-DTPA enhancement in DCE-MRI scans may assist development of a clinically robust (i.e., unambiguous) technique for PDT outcome assessment.

Monitoring Pc 4-mediated Photodynamic Therapy of U87 Tumors with Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI) in the Athymic Nude Rat

Post-operative verification of the specificity and sensitivity of photodynamic therapy (PDT) is most pressing for deeply placed lesions such as brain tumors. We wish to determine whether Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI) can provide a non-invasive and unambiguous quantitative measure of the specificity and sensitivity of brain tumor PDT. Methods: 2.5 x 105 U87 cells were injected into the brains of six athymic nude rats. After 5-6 days, the animals received 0.5 mg/kg b.w. of the phthalocyanine photosensitizer Pc 4 via tail-vein injection. On day 7 peri-tumor DCE-MRI images were acquired on a 7T microMRI scanner before and after tail-vein administration of 100 μL gadolinium and 400 μL saline. After this scan the animals received a 30 J/cm2 dose of 672-nm light from a diode laser (i.e., PDT). The DCE-MRI scan protocol was repeated on day 13. Next, the animals were euthanized and their brains were explanted for Hematoxylin and Eosin (H&E) histology. Results: No tumor was found in one animal. The DCE-MRI images of the other five animals demonstrated significant tumor enhancement increase (p < 0.053 two-sided t-test and p < 0.026 one-sided t-test) following PDT. H&E histology presented moderate to severe tumor necrosis. Discussion: The change in signal detected by DCE-MRI appears to be due to PDT-induced tumor necrosis. This DCE-MRI signal appears to provide a quantitative, non-invasive measure of the outcome of PDT in this animal model and may be useful for determining the safety and effectiveness of PDT in deeply placed tumors (e.g., glioma).

Use of optical pharmacokinetics systems (OPS) for non-invasive measurement of Phthalocyanine 4 (Pc 4) concentrations in mice bearing MDA-MB-231 xenografts

Objective: Pc 4, a phthalocyanine photosensitizer in Phase I photodynamic therapy (PDT) trials, requires laser activation near 672 nm. For effective PDT, photosensitizer must be present in the target tissues. OPS uses elastic scattering spectroscopy to measure Pc 4 optical absorption non-invasively, and that absorbance can be converted to concentration using Pc 4 standard curves in 1% Intralipid®. In this study, we used OPS to evaluate Pc 4 optical absorption with time in subcutaneous tumor (with or without laser activation) and in contralateral skin. Tumor response was also evaluated after Pc 4-PDT. Conclusions: Both Pc 4 and hemoglobin optical absorption could be monitored by OPS. The decrease of Pc 4 absorption after PDT and the appearance of d-hbg indicated that alterations occurred in the tumor following Pc 4-PDT. The increase in d-hbg suggests that oxygen was not replaced completely, possibly due to circulation damage in tumor.

Apoptosis mechanisms related to the increased sensitivity of Jurkat T-cells versus A431 epidermoid cells to photodynamic therapy with the phthalocyanine Pc 4 (Photochemistry and Photobiology (2008) 84, 2, (407-411))

Figure 4. Sensitivity to Pc 4-PDT. (a) Example of a cell cytotoxicity study using the MTT assay. Cells of each line were plated in 96-well plates, exposed to a series of Pc 4 concentrations followed by red light at 200 mJ ⁄ cm, and returned to the 37 C incubator for 24 hours. Cytotoxicity was assayed with MTT, and the LD50 determined (in this case, 20 nM for Jurkat cells and 140 nM for A431 cells). (b) Plot of LD50 of Pc 4 plotted versus fluence, for experiments on A431 (diamonds) and Jurkat (squares) cell lines. Solid and dashed lines are least-squares regression lines obtained by regressing Ln(LD50) vs Ln(Fluence) for A431 and Jurkat cells, respectively. (c) Quantification of Pc 4-PDT induced apoptosis. A431 cells (left) and Jurkat cells (right) were incubated with Pc 4-loaded growth medium for two h, followed by irradiation with 200 mJ ⁄ cm red light. After further incubation for 4 (open bars) or 18 (hatched bars) h, cells were fixed in 3.7% formaldehyde for 30 min at room temperature, followed by staining with Hoechst 33342 (Molecular Probes, Eugene, OR) for 30 min at room temperature. Slides were viewed under a fluorescence microscope. Cells displaying morphological apoptosis were quantified and normalized to untreated control cells. Data represent an average of 2–3 independent experiments ± standard error or range, except for A431 cells at 100 nM Pc 4, which are from single experiments.