Biological validation of Lumiblast in an orthotopic mouse brain tumor model
1. Purpose: Photodynamic therapy (PDT) is a treatment that uses a drug, called a photosensitizer and a particular wavelength of external light. When photosensitizers are exposed to a specific wavelength of light, they produce a form of oxygen that kills nearby cells. The main limitation of PDT is the access of light into the deep cancerous lesions. In Lumiblast project (FOTS # 21339), Luminol (a forensic tool) provides the energy required for metabolization of photosensitizer. The compromised blood brain barrier leads to accumulation of photosensitizer and Luminols at the GBM regions. Most tumours exhibit due to their more primitive mitochondria and cancer cells have long been observed to have an increased production of ROS relative to normal cells and in particular formation of ROS in the mitochondria. In Lumiblast, we modified the chemiluminescent dyes so that they exhibit a high affinity to cell mitochondria. As a result the activation of the chemiluminescent moieties in these cells is expected to be much higher than in normal cells. In Lumiblast, mitotropic luminol is expected to employ the reactive oxygen species (ROS) produced by the electrons leaking from the electron transport chain (ETC) and reduce molecular oxygen to superoxide anion and then hydrogen peroxide, to produce luminescence which provides the energy for metabolization of photosensitizer. Photosensitizer-based fluorescence produces singlet oxygen that can wipe out the Glioblastoma (GBM) cells. We tested Lumiblast in vitro and in heterotopic models of GBM (subcutaneous implantation of GBM cells). Lumiblast reduced cell viability and tumor growth rate in in vitro and heterotopic models, respectively. Orthotopic model represents the final stage in preclinical cancer research. In this pilot study, we will set up the method in our lab for documenting the effect of Lumiblast in intracranially located GMB tumors.
2. Harmful effects: Stereotaxic surgery for intracranial implantation of cancer cells and subcutaneous implantation of osmotic pump for drug delivery has short-term mild to moderate discomfort and pain. Luminols and photosensitizer may induce short-term mild to moderate distress and reduction in body weight. Tumor growth may have neuorological symptoms, like seizures and ataxia.
3. Expected utility value: There is no curative treatment method for glioblastoma and there is a short lifespan after diagnosis. Accordingly, the possible usefulness of this treatment will be significant as this cancer is mainly incurable.
4. Number of animals: 340 NSG female mice.
5. 3R: The number of animals as dictated by our previous (xenograft) experiments (FOTS #21339), literature and requirements for statistical significance. We considered the minimum number of mice in all groups which provide statistically acceptable results. A number of compounds have been synthesized that generate luminescence. They were tested on cells, and 3 most effective ones which have been tested in subcutaneous model will be used in orthotopic model. Some parts of controls will be reused or eliminated along the way if they do not provide important additional information. The aim of this experiment may not be accomplished using alternative methodsThe methods have been and will be refined to avoid pain and distress
2. Harmful effects: Stereotaxic surgery for intracranial implantation of cancer cells and subcutaneous implantation of osmotic pump for drug delivery has short-term mild to moderate discomfort and pain. Luminols and photosensitizer may induce short-term mild to moderate distress and reduction in body weight. Tumor growth may have neuorological symptoms, like seizures and ataxia.
3. Expected utility value: There is no curative treatment method for glioblastoma and there is a short lifespan after diagnosis. Accordingly, the possible usefulness of this treatment will be significant as this cancer is mainly incurable.
4. Number of animals: 340 NSG female mice.
5. 3R: The number of animals as dictated by our previous (xenograft) experiments (FOTS #21339), literature and requirements for statistical significance. We considered the minimum number of mice in all groups which provide statistically acceptable results. A number of compounds have been synthesized that generate luminescence. They were tested on cells, and 3 most effective ones which have been tested in subcutaneous model will be used in orthotopic model. Some parts of controls will be reused or eliminated along the way if they do not provide important additional information. The aim of this experiment may not be accomplished using alternative methodsThe methods have been and will be refined to avoid pain and distress