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.

Photodynamic therapy is a promising cancer treatment that employs wavelength-specific light in combination with a photosensitizing agent to induce local tumor destruction by photochemical generation of cytotoxic singlet oxygen. Clinical PDT has been evaluated for a variety of primary tumors, however, its use in spinal metastases to our knowledge has not been previously evaluated. A practical consideration is the ability to deliver light to bone. The investigators are evaluating a novel method of applying light to targeted spinal lesions using a minimally invasive technique similar to percutaneous vertebroplasty. This preliminary preclinical study evaluates the feasibility and efficacy of spinal PDT.

To evaluate the feasibility and efficacy of spinal meta-static photodynamic therapy (PDT) using a percutaneous minimally invasive surgical approach similar to that of vertebroplasty in a preclinical model of bone metastases.

A bioluminescent metastatic model was developed (intracardiac injection 2x10⁶ MT-1^Luc human breast cancer cells; rnu/rnu rats). In forty-three tumor bearing rats, a PDT light dose escalation trial (photosensitizer BPD-MA;2mg/Kg IV) was conducted to assess safety and efficacy of tumor ablation in a single treatment via an implanted optical fibre held adjacent to targeted spinal lesions. Pre and forty-eight hours post bioluminescent imaging was performed to gauge PDT related effects followed by post-sacrifice microCT and histology.

Spinal PDT caused a reduction in bioluminescence of targeted lesions (66% to 87% in three hour drug-light group using light fluence rates of 25J and 150J, respectively; p< 0.05). The most selective drug-light interval was twenty-four hours where PDT induced tumor cell apoptosis/necrosis occurred, however, no spinal cord injury was observed. The greatest anti-tumor effect was observed at the three hour drug-light interval but observations of neurologic sequalae (9/22 animals) highlight the importance of ongoing study to closely define the therapeutic window of PDT.

Drug dosimetry and the drug-light interval are critical in establishing an efficacious and safe treatment range for spinal PDT. Bioluminescent reporter imaging provides an in vivo longitudinal assessment of tumor growth kinetics. The feasibility of the minimally invasive approach for spinal PDT in this model has been established.

Funding: this study was support, in part, by a CBCRA (formerly CBCRI) Idea’s grant

Purpose: There is a clinical need for novel effective local therapies to treat spinal metastases at significant risk for fracture. Photodynamic therapy (PDT) is a promising cancer treatment that employs wavelength specific light combined with a photosensitizing agent to induce localized tumour destruction by photochemical generation of singlet oxygen. Using minimally invasive techniques developed for vertebroplasty to deliver light within the vertebral body, PDT is proposed as a potential new treatment for spinal metastases; however, the effects of PDT on bone are largely unknown. This study aims to determine if PDT affects the structural integrity of normal vertebral bone.

Methods: Sixteen Wistar rats were randomly assigned to control, 1-week treatment or 6-week treatment groups. Rats treated with PDT received an intracardiac injection of 2mg/kg BPD-MA activated at 15 minutes post-injection through administration of a non-thermal 690nm diode laser positioned adjacent to the L3 vertebral body via fluoroscopic guidance (150J at 150mW). Rats were sacrificed at 1-week or 6-weeks following a single treatment. |In vitro & #956;CT scans were taken of L2-L4 and 3D stereological quantities measured using a semiautomated volume shrinkage thresholding technique within the trabecular bone centrum. L2, L3 and L4 vertebral bodies were individually tested biomechanically to failure in axial compression. Yield stress and stiffness were calculated from generated load displacement curves.

Results: Bone surface area and bone volume significantly increased with treatment, through trabecular thickening, at both 1-week and 6-weeks vs. control group. The treated group demonstrated an increase in yield stress at 6-weeks vs. control (27%, p=0.023). An increase in stiffness (45%, p=0.010) was found in the 1-week treatment group vs. control, but was not maintained in the 6-week group.

Conclusions: PDT is a promising new treatment for spinal metastases that appears to strengthen vertebral bone. Further research must determine the mechanism of this action and verify if similar effects will occur in metastatically-involved vertebrae. If PDT proves to be effective in both destroying tumour cells and in strengthening remaining bone, it may provide a very attractive minimally invasive treatment option for patients with spinal metastases.

Funding : Other Education Grant

Funding Parties : Canadian Breast Cancer Foundation, Ontario Chapter

Purpose: This study investigates the use of photodynamic therapy (PDT) in regulating bone development with a view to its potential role in treating Juvenile leg length discrepancy (LLD).

Methods: Transgenic mice expressing the luciferase firefly gene upon activation of a promoter sequence specific to the vascular endothelial growth factor (VEGF) gene were subject to benzoporphyrin derivative monoacid (BPD-MA)-mediated PDT in the right, tibial epiphyseal growth plate at the age of 3 weeks. BPD-MA was administered intracardially (2mg/kg) followed 10 mins later by a laser light (690 +/− 5 nm) at a range of doses (5-27J, 50 mW output) delivered either as a single or repeat regimen (x2-3). Contra-lateral legs served as no-light controls. Further controls included animals that received light treatment in the absence of photosensitizer or no treatment. Mice were imaged for VEGF related bioluminescence (photons/sec/steradian) at t= 0, 24, 48, 72 h and 1–4 weeks post PDT. FaxitronÔ x-ray images provided accurate assessment of bone morphometry. Upon sacrifice, the tibia and femur of the treated and untreated limbs were harvested, imaged and measured again and prepared for histology. A number of animals were sacrificed at 24 h post PDT to allow immunohistochemical staining for CD31, VEGF and hypoxia-inducible factor (HIF-1 alpha) within the bone.

Results: PDT-treated (10 J, x2) mice displayed enhanced bioluminescence at the treatment site (and ear nick) for up to 4 weeks post treatment while control mice were bioluminescent at the ear-nick site only. Repeat regimens provided greater shortening of the limb than the corresponding single treatment. PDT-treated limbs were shorter by 3–4 mm on average as compared to the contra lateral and light only controls (10 J, x2). Immunohistochemistry confirmed the enhanced expression VEGF and CD31 at 4 weeks post-treatment although no increase in HIF-1& #945; was evident at either 24 h or 4 weeks post PDT treatment.

Conclusions: Results confirm the utility of PDT to provide localized effects on bone development that may be applicable to other related skeletal deformities.

Photodynamic therapy (PDT) is a promising new treatment for spinal metastases; however, the effects of PDT on bone are largely unknown. This study assessed the impact of PDT on spinal stability in rats at high (non-therapeutic) drug and LASER light doses. Spinal stability was assessed using stereological measures attained from in vitro μCT scans. High doses of PDT were shown to cause a reduction in vertebral density. Postoperative paralysis was also noted in a subset of animals treated. Tumour-involved vertebrae are already mechanically weakened; as such it is essential to establish a safe and efficacious therapeutic window for vertebral PDT.

This study assessed the effect of high doses of photodynamic therapy (PDT) on biomechanical stability and bone density of lumbar vertebrae.

PDT can cause damage to the vertebral bone and induce paralysis when treatment is applied at very high doses in the rat spine.

PDT is a promising new treatment for spinal metastases however, it is important to understand its effect on vertebral bone in order to closely define the therapeutic window for safety and efficacy.

Trabecular bone density decreased from L1–L3 in normal, untreated rats. The L2 vertebra when treated with high dose PDT was shown to have decreased bone density as compared to both L1 and L3. As expected, tumour-bearing rats had lower vertebral densities than normals.

Rnu/Rnu rats were separated into normal controls, normals treated with PDT and tumour-bearing rats. Rats treated with PDT received an intercardiac injection of 2.5mg/Kg BPD-MA. The drug was activated through administration of 500J (300mA) of a non-thermal 690nm LASER adjacent to the L2 vertebral body. After one week, in vitro μCT scans were taken of L1–L3 and stereological quantities measured.

The demonstrated reduction of bone density as quantified one week following treatment is important when considering spinal stability in the potential use of PDT to treat vertebral metastases. We have observed that the therapy can induce paralysis when treatment is applied at high doses in the rat spine. The intermediate and long-term effects of PDT on bone remain unknown and require ongoing study.