Quantification of Protoporphyrin IX Accumulation in Glioblastoma Cells: A New Technique

Abstract

Introduction. 5-Aminolevulinic Acid (5-ALA) is a precursor of heme synthesis. A metabolite, protoporphyrin IX (PpIX), selectively accumulates in neoplastic tissue including glioblastoma. Presurgical administration of 5-ALA forms the basis of fluorescence-guided resection (FGR) of glioblastoma (GBM) tumors. However, not all gliomas accumulate sufficient quantities of PpIX to fluoresce, thus limiting the utility of FGR. We therefore developed an assay to determine cellular and pharmacological factors that impact PpIX fluorescence in GBM. This assay takes advantage of a GBM cell line engineered to express yellow fluorescent protein. Methods. The human GBM cell line U87MG was transfected with a YFP expression vector. After treatment with a series of 5-ALA doses, both PpIX and YFP fluorescence were measured. The ratio of PpIX to YFP fluorescence was calculated. Results. YFP fluorescence permitted the quantification of cell numbers and did not interfere with 5-ALA metabolism. The PpIX/YFP fluorescence ratio provided accurate relative PpIX levels, allowing for the assessment of PpIX accumulation in tissue. Conclusion. Constitutive YFP expression strongly correlates with cell number and permits PpIX quantification. Absolute PpIX fluorescence alone does not provide information regarding PpIX accumulation within the cells. Our research indicates that our PpIX/YFP ratio assay may be a promising model for in vitro 5-ALA testing and its interactions with other compounds during FGR surgery.

Small molecule additive enhances cell uptake of 5-aminolevulinic acid and conversion to protoporphyrin IX

“…CHO-K1 cells were chosen as a convenient model system for this study because the kinetics of PpIX production are well characterized. 39 In agreement with studies using other cell lines, 28,40 intracellular PpIX production reached a plateau when the 5-ALA concentration was > ~800 μM (Figure 2). To ensure that any additive enhancement effect was not masked by this PpIX plateau, a standard set of cell treatment conditions was employed (100 μM 5-ALA and 6 hour incubation time) for all experiments.…”

Alterations in Cell Motility, Proliferation, and Metabolism in Novel Models of Acquired Temozolomide Resistant Glioblastoma

“…The top hit from GC-TOF-MS was the non-aminogenic acid aminomalonate, an inhibitor of aminolevulinic acid synthase, the enzyme that catalyzes the rate limiting step of the protoporphyrin IX(PpI IX)/heme synthesis pathway 16 . This pathway is deregulated in many cancers, including GBM, and can be exploited to identify tumor area during surgery 17,18 . The last significant metabolite was citric acid, which has been shown to decrease overall survival in GBM patients with higher citric acid in their cerebral spinal fluid.…”

Integrating clinical, molecular, proteomic and histopathological data within the tissue context: tissunomics

“…During tumour progression and after chemotherapy or radiotherapy, genetic alterations accumulate to select for the best‐adapted clone. Molecular heterogeneity has been described in renal adenocarcinoma, breast cancer and glioblastoma, among others . It is thought that these clones with greater proliferative advantages and resistance to local stresses then predominate …”

“…Molecular heterogeneity has been described in renal adenocarcinoma, breast cancer and glioblastoma, among others. 19 It is thought that these clones with greater proliferative advantages and resistance to local stresses then predominate. [20][21][22][23][24] Other theories include the tissue organisation field theory (TOFT), which postulates that neoplasia arises from a local tissue disorder and may be due to a defect in interaction between cells and other tissue components, 15,[25][26][27] where epigenetic or genetic changes affect both epithelial and stromal cells in multiple locations.…”