Microbubble-mediated Focused Ultrasound in Diffuse Midline Glioma H3 K27M

Abstract

Life expectancy of children diagnosed with cancer has increased in the past few decades. This is illustrated by a five-year survival of nearly 80 % for most cancers. However, this has not been observed for pediatric brain tumors which account for 40 % of the cancer-related deaths in children. The most fatal pediatric brain cancer is high-grade glioma (HGG) with an overall survival of less than 5 %. The HGG subtype diffuse midline glioma (DMG), H3 K27M localized in the midline structure of the brain, has one of the worst prognoses with a median survival of only 11 months. Resection of these tumors is usually not possible due to their location. DMG H3 K27M is located in the midline structures such as brainstem and thalamus which are important for vital functions since it regulates for example cardiac function, respi- ratory function, and sleep. Radiotherapy, albeit palliative, is the only method that can relive symptoms for a short period of time. Conventional chemotherapy has not prolonged sur- vival since the efficacy of chemotherapeutics is low, at least in part due to the presence of the blood-brain barrier (BBB). This protective barrier prevents drugs from entering the brain parenchyma and actively exports drugs back into the bloodstream. In order to circumvent the BBB, alternative drug delivery methods have been proposed that deliver drugs into the brain tumor. In Chapter 2 we discuss the use of nanoparticles, microbubble-mediated fo- cused ultrasound, convection enhanced delivery (CED), intranasal delivery, and intra-arterial delivery to increase the concentration of drugs in the brain. These drug delivery methods have been used in (pre)clinical trials with various results. DMG H3 K27M that resides in the brainstem requires a non-invasive method to circumvent the BBB since the brainstem is a delicate structure regulating vital functions. Microbub- ble-mediated focused ultrasound is such a non-invasive method that can specifically target the tumor area for local drug delivery into the brain tumor. Upon the application of ultra- sound waves, microbubbles start to oscillate against the endothelial cell wall of blood ves- sels. This initiates transcytosis and disruption of the tight junctions between the endothelial cells, allowing for paracellular transport of drugs. Microbubble-mediated focused ultrasound is a reversible process because within several hours the BBB returns to its original state. In Chapter 3, we describe the in-house built focused ultrasound system for high-throughput small animal studies. Here, we used bioluminescence (BLI) and X-ray to guide the trans- ducer to the brain tumor. Microbubble vibrations, an indication of safety, were monitored with integrative cavitation detection monitoring. The system has been validated for DMG H3 K27M mouse models. We used this system for the treatment of DMG H3 K27M xeno- graft mice, described in Chapter 4. Previous studies showed that single exposure of su- pratentorial tumors to doxorubicin exhibited a prolonged overall survival. However, other research showed that DMG H3 K27M tumors treated with microbubble-mediated focused ultrasound in combination with doxorubicin did not result in a survival benefit in a xenograft DMG H3 K27M mouse model. Hence, we hypothesized that the duration of exposure for doxorubicin was too short. Using liposomal formulations of doxorubicin (Caelyx® and 2B3- 101) that slowly release doxorubicin over time, we could potentially prolong exposure of doxorubicin to the brain tumor. DMG H3 K27M xenograft models were established through orthotopic injections of HSJD-DIPG-07 tumor cells into the pontine area of female athymic nude-foxn1nu mice. Tumor engraftment was confirmed with BLI. Using the in-house built focused ultrasound system, we treated mice with 5 mg/kg 2B3-101 or Caelyx® 1 h before microbubble-mediated focused ultrasound or 5 mg/kg free doxorubicin immediately after microbubble-mediated focused ultrasound. Mice were regularly monitored until humane endpoint was reached. After statistical analysis, we did, however, not observe a significant improvement in survival. In Chapter 5 we studied the neurovascular unit (NVU) in DMG H3 K27M patients. The NVU is a functional unit consisting of the BBB, neurons and perivascular microglia. Pre-treatment biopsy samples were obtained from Princess Máxima Center for Pediatric Oncology (Utrecht, The Netherlands), end-stage disease DMG H3 K27M autopsy samples were obtained from Amsterdam UMC, location VUmc (Amsterdam, The Netherlands), and age-matched healthy pontine tissue samples were obtained from NIH NeuroBioBank (Maryland, United States). Tissue was stained for BBB markers claudin-5, ZO-1, laminin, PDGFR-β and efflux transporter P-gp. Expression of claudin-5, ZO-1, laminin, PDGFR-β, and P-gp was reduced in both au- topsy and biopsy samples compared to healthy pontine tissue. Furthermore, the vascular density was significantly lower in DMG H3 K27M autopsy samples compared to the healthy pons whereas the median vascular diameter was not significantly different. To determine if these structural changes in the NVU of DMG H3 K27M patients were also present in the DMG H3 K27M animal models we used in preclinical studies, we investigated the state of the BBB after tumor engraftment in mice. In Chapter 6 we describe the BBB markers in different xenograft mouse models. We observed differences in BBB marker expression, since this is a pilot study more research is needed to determine if these differences are indeed due to the tumor model. We conclude this thesis with a discussion, Chapter 7, of the results and findings from our studies