Purpose: Bony metastases in vertebrae secondary to breast cancer can result in osteolysis and an increase in skeletal related events. Bisphosphonates (BP) are the current standard of care for breast cancer patients with skeletal disease. Photodynamic therapy (PDT) is a non-radiative treatment, which has been successfully applied to various malignancies and shown to successfully ablate vertebral human breast cancer (MT1) metastases in a murine model. Previous in-vitro study has shown that pre-treatment of MT-1 cells with the BP zoledronic acid (Zometa^®) renders them more susceptible to PDT. The aim of this study was to evaluate the influence of pre-treatment with BPs on the effect of PDT treatment on tumour ablation in metastatically involved vertebrae in vivo.

Method: Metastases were induced in fourteen 5–6 weeks old female athymic rats (Hsd:RH-Foxn1rnu) by intra-cardiac injection of 2x10^6 MT-1 cells. Four groups were formed:

control, no treatment;

Seven days after MT-1 injection 60 μg/kg of zoledronic acid was injected. PDT treatment was administered on day 14 using the photosensitizer BPD-MA (1.0 mg/kg; Visudyne). Fifteen minutes later, laser-light (690nm; 75J) was administered to the lumbar vertebrae. The rats were euthanized 7 days after PDT treatment. A total of 45 vertebrae were evaluated using a histomorphometric program (GENIE™, Aperio) to assess tumour burden. Statistical analyses were performed using a one-way ANOVA with a Tukey post hoc test. A p-value p< .05 was considered to be statistically significant..

Results: The total The total tumour burden within vertebrae of rats pre-treated with BP and/or PDT was significantly lower compared to the control rats (p< .001). In addition, the PDT alone treated group demonstrated significantly less tumour burden than the combined BP+PDT group. In the control and BP-only groups, large tumours were found to include regions of necrosis. The PDT treatment groups (PDT and BP+PDT) exhibited areas of necrosis throughout the entire vertebral bodies with adjacent formation of granulation tissue.

Conclusion: BP, PDT and combined BP+PDT treatments resulted in a lower overall tumour burden at day 21 post MT-1 cell injection compared to control rats. A surprising increased level of tumour burden was found in comparing the combined treatment group to the PDT-only group. These findings are in contrast to previous in-vitro results, where the pre-treatment with BPs made the cells more susceptible to PDT. Pre-treatment with BP affects both the bone and tumour cells, and as such may induce different cellular pathways in response to PDT treatment. However, the ability of PDT applied at day 14 to cause a similar reduction in tumour burden compared to BP treatment at day 7, suggests its ability to rapidly and effectively ablate the tumour within the bone, even in the presence of BP.

Bone is the preferred site of metastases in women with breast cancer, which can cause skeletal-related events (SRE¡¦s) such as pathologic fractures. Bisphosphonates are the current standard of care for treatment of meta-static bone disease by preventing further bone destruction. Photodynamic therapy (PDT) has been applied successfully as a non-radiative treatment for malignancies. In PDT, light is delivered to a tumour after the administration of a photosensitiser. Earlier pre-clinical studies in a metastatic rat model have shown that PDT reduced the tumour burden in the vertebrae. The goal of this investigation was to study the effect of PDT on bisphosphonate pre-treated cancer in-vitro.

Human breast cancer cells, MT-1, were cultured until confluent. The following groups were formed: no treatment; incubation with zoledronic acid (24h; 10 ƒÝmol) only; PDT treatment only and incubation with zoledronic acid and PDT treatment. Prior to light application 1 microg/ml of the photosensitiser BPD-MA was added. PDT was performed with a light dose of 1J and 10 J. The cells were stained with a live/dead stain and analyzed by fluorescence microscope and flowcytometry.

Incubation of the MT-1 carcinoma cells with bisphosphonate zoledronic acid resulted in a significantly higher number of dying cells following PDT treatment when compared cells that were not treated by zoledronic acid (p< 0.05). When comparing cell groups that did not undergo PDT treatment the incubation with zoledronic acid alone did not have a statistically significant effect on cell survival twenty-four hours following zoledronic acid administration.

In-vitro, breast cancer cells appear more susceptible to PDT after they have been incubated with the zoledronic acid. Zoledronic acid, a potent bisphosphonate, inhibits farsenylpyrophosphate (FPP) which is involved in farsenylation of cell membrane proteins. The inhibition of FPP may cause a reduced effect of PDT on cell rescue. The treatment with bisphosphonates seems to have a synergistic effect with PDT treatment. As such, light dosimetry in PDT treatment may need to take into account potential therapeutic interactions between PDT and current medical therapies in the treatment of skeletal metastatic burden.

The purpose of the study was to examine the effects of vascular-targeted photodynamic therapy (PDT) using benzoporhyrin derivative (BPD) on growth plates in spine and long bones. Specifically we wish to determine whether the ipsilateral up-regulation of VEGF in the thoracic and/or lumbar spine following treatment with leads to onset of scoliosis morphologically similar to idiopathic adolescent scoliosis. And secondly confirm growth plate closure in long bones following BPD-PDT resulting in leg length discrepancy.

A 0.2 mm fiber was placed through an 18g needle onto one side of the distal femoral epiphysis (n=24) or lower thoracic/upper lumbar vertebral bodies of four-week old mice (n=18). Mice are genetically modified to emit bioluminescence upon activation of the vascular endothelial growth factor gene (VEGF). Accurate placement was confirmed using fluoroscopy. BPD (2 mg/Kg, i.v.) was administered systemically and the growth plates were stimulated with 690nm laser light five minutes later. Range of light dose regimens were tested. Animals were followed for a total of seven-twelve weeks post treatment. Faxitron imaging and bioluminescent imaging were obtained to determine leg length or curve progression and VEGF activity. Histology and immunohistochemistry including H& E, HIF-1á, CD31 and VEGF immunohistochemistry was performed.

PDT was able to up-regulate VEGF for up to four weeks following treatment following a percutaneous treatment using a 0.2mm treatment fiber both in the femur and vertebrae. Femoral shortening occurred with histological evidence of bone formation across the growth plate. We were able to identify using faxitron abnormal curvature in a number of the animals that received 5J, 10 mW regimen.

This study confirms that that the epiphyses of vertebrae and long bones are similarly susceptible to the effects of a hypoxic insult resulting in VEGF up-regulation. We are proposing that this stress response can lead to premature closure of epiphyseal growth plates of long bones resulting in limb growth arrest or asymmetric growth of vertebrae and the development of scoliosis in an animal model.