The neurovascular unit in diffuse intrinsic pontine gliomas

Aims: Diffuse intrinsic pontine glioma (DIPG) is a childhood brainstem tumor with a median overall survival of eleven months. Lack of chemotherapy efficacy may be related to an intact blood-brain barrier (BBB). In this study we aim to investigate the neurovascular unit (NVU) in DIPG patients. Methods: DIPG biopsy (n = 4) and autopsy samples (n = 6) and age-matched healthy pons samples (n = 20) were immunohistochemically investigated for plasma protein extravasation, and the expression of tight junction proteins claudin-5 and zonula occludens-1 (ZO-1), basement membrane component laminin, pericyte marker PDGFR-β, and efflux transporters P-gp and BCRP. The mean vascular density and diameter were also assessed. Results: DIPGs show a heterogeneity in cell morphology and evidence of BBB leakage. Both in tumor biopsy and autopsy samples, expression of claudin-5, ZO-1, laminin, PDGFR-β and P-gp was reduced compared to healthy pontine tissues. In DIPG autopsy samples, vascular density was lower compared to healthy pons. The density of small vessels (<10 µm) was significantly lower (P<0.001), whereas the density of large vessels (≥10 µm) did not differ between groups (P = 0.404). The median vascular diameter was not significantly different: 6.21 µm in DIPG autopsy samples (range 2.25-94.85 µm), and 6.26 µm in controls (range 1.17-264.77 µm). Conclusion: Our study demonstrates evidence of structural changes in the NVU in DIPG patients, both in biopsy and autopsy samples, as well as a reduced vascular density in end-stage disease. Adding such a biological perspective may help to better direct future treatment choices for DIPG patients

Microbubble-mediated Focused Ultrasound in Diffuse Midline Glioma H3 K27M

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

Overview of Current Drug Delivery Methods Across the Blood–Brain Barrier for the Treatment of Primary Brain Tumors

Existing drug delivery methods have not led to a significant increase in survival for patients with malignant primary brain tumors. While the combination of conventional therapies consisting of surgery, radiotherapy, and chemotherapy has improved survival for some types of brain tumors (e.g., WNT medulloblastoma), other types of brain tumors (e.g., glioblastoma and diffuse midline glioma) still have a poor prognosis. The reason for the differences in response can be largely attributed to the blood–brain barrier (BBB), a specialized structure at the microvasculature level that regulates the transport of molecules across the blood vessels into the brain parenchyma. This structure hampers the delivery of most chemotherapeutic agents for the treatment of primary brain tumors. Several drug delivery methods such as nanoparticles, convection enhanced delivery, focused ultrasound, intranasal delivery, and intra-arterial delivery have been developed to overcome the BBB in primary brain tumors. However, prognosis of most primary brain tumors still remains poor. The heterogeneity of the BBB in primary brain tumors and the distinct vasculature of tumors make it difficult to design a drug delivery method that targets the entire tumor. Drug delivery methods that combine strategies such as focused ultrasound and nanoparticles might be a more successful approach. However, more research is needed to optimize and develop new drug delivery techniques to improve survival of patients with primary brain tumors

A high-throughput image-guided stereotactic neuronavigation and focused ultrasound system for blood-brain barrier opening in rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (>500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma. In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs. Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma