The current study reports that KetoCal® (KC; 4:1 fat:protein/carbohydrates), fed ad libitum, alters hypoxia, angiogenic, and inflammatory pathways in a mouse model of glioma. Tumors from animals maintained on KC showed reduced expression of the hypoxia marker carbonic anhydrase 9 (CA IX), a reduction in hypoxia inducible factor 1-alpha (HIF-1α) and decreased activation of nuclear factor kappa B (NF-κB). Animals maintained on KC also showed a reduction in expression of vascular endothelial growth factor receptor 2 (VEGFR2) and decreased microvasculature in their tumors. Further, peritumoral edema was significantly reduced in animals fed the KC and protein analysis showed significantly altered expression of the tight junction protein zona occludens-1 (ZO-1) and the water channeling protein aquaporin-4 (AQP4), both of which have been implicated in malignant processes in glioma, including the formation of peritumoral edema in patients. Taken together the data suggests that KC alters multiple processes involved in malignant progression of gliomas. A greater understanding of the effects of the ketogenic diet as an adjuvant therapy will allow for a more rational approach to its clinical use.
This thesis scrutinizes CLE technology for its ability to provide real-time intraoperative in vivo and ex vivo visualization of histopathological features of the normal and tumor brain tissues. First, the optimal settings for CLE imaging are studied in an animal model along with a generational comparison of CLE performance. Second, the ability of CLE to discriminate uninjured normal brain, injured normal brain and tumor tissues is demonstrated. Third, CLE was used to investigate cerebral microvasculature and blood flow in normal and pathological conditions. Fourth, the feasibility of CLE for providing optical biopsies of brain tumors was established during the fluorescence-guided neurosurgical procedures. This study established the optimal workflow and confirmed the high specificity of the CLE optical biopsies. Fifth, the feasibility of CLE was established for endoscopic endonasal approaches and interrogation of pituitary tumor tissue. Finally, improved and prolonged near wide-field fluorescent visualization of brain tumor margins was demonstrated with a scanning fiber endoscopy and 5-aminolevulinic acid.
These studies suggested a novel paradigm for neurosurgery-pathology workflow when the noninvasive intraoperative optical biopsies are used to interrogate the tissue and augment intraoperative decision making. Such optical biopsies could shorten the time for obtaining preliminary information on the histological composition of the tissue of interest and may lead to improved diagnostics and tumor resection. This work establishes a basis for future in vivo optical biopsy use in neurosurgery and planning of patient-related outcome studies. Future studies would lead to refinement and development of new confocal scanning technologies making noninvasive optical biopsy faster, convenient and more accurate.
A major hindrance to advances in the care of patients with malignant gliomas is the presence of the blood brain barrier (BBB) and blood-brain tumor barrier (BBTB) that greatly restricts drug access from the plasma to the tumor cells. Bubble-assisted Focused Ultrasound (BAFUS) has proven effective in opening the BBB for treatment of glial tumors in adults and pediatric cases. BAFUS has been previously shown to disrupt noninvasively, selectively, and transiently the BBB in small animals in vivo. However, there is a lack of an in vitro preclinical model suitable for testing the genetic determinants of endothelial cell tight junction integrity and vulnerability to the physical disruption. Our BBB organ-on-chip platform will enable precision medicine of brain cancers through identifying patient-specific parameters by which to open the BBB allowing use of drugs and drug combinations otherwise unsuitable. We intend to sequence these in vitro models to verify that the genotype (alleles/SNPs) of tight junction proteins contribute to BBB structure and integrity. To initiate this effort, we report the development of an ultrasound transparent organ-on-chip model populated by iPSC-derived endothelial cells (iPSC-EC) co-cultured with astrocytes. Western blot, immunocytochemistry, and transelectrical endothelial resistance (TEER) studies all convey expression of key EC proteins and marked barrier integrity. Successful iPSC differentiation, tight junction formation, and annotation of tight junction alleles will be presented. Efforts are underway to benchmark device-ultrasound interactions, disruption vulnerability, and determine associations between iPSC-EC genotype and phenotype.