Assessment of Cannabidiol and ∆9-Tetrahydrocannabiol in Mouse Models of Medulloblastoma and Ependymoma

Children with medulloblastoma and ependymoma are treated with a multidisciplinary approach that incorporates surgery, radiotherapy, and chemotherapy; however, overall survival rates for patients with high-risk disease remain unsatisfactory. Data indicate that plant-derived cannabinoids are effective against adult glioblastoma; however, preclinical evidence supporting their use in pediatric brain cancers is lacking. Here we investigated the potential role for ∆9- tetrahydrocannabinol (THC) and cannabidiol (CBD) in medulloblastoma and ependymoma. Dose- dependent cytotoxicity of medulloblastoma and ependymoma cells was induced by THC and CBD in vitro, and a synergistic reduction in viability was observed when both drugs were combined. Mechanistically, cannabinoids induced cell cycle arrest, in part by the production of reactive oxygen species, autophagy, and apoptosis; however, this did not translate to increased survival in orthotopic transplant models despite being well tolerated. We also tested the combination of cannabinoids with the medulloblastoma drug cyclophosphamide, and despite some in vitro synergism, no survival advantage was observed in vivo. Consequently, clinical benefit from the use of cannabinoids in the treatment of high-grade medulloblastoma and ependymoma is expected to be limited. This study emphasizes the importance of preclinical models in validating therapeutic agent efficacy prior to clinical trials, ensuring that enrolled patients are afforded the most promising therapies available

Conventional therapies deplete brain-Infiltrating adaptive immune cells in a mouse model of group 3 medulloblastoma implicating myeloid cells as favorable immunotherapy targets

Medulloblastoma is the most common childhood brain cancer. Mainstay treatments of radiation and chemotherapy have not changed in decades and new treatment approaches are crucial for the improvement of clinical outcomes. To date, immunotherapies for medulloblastoma have been unsuccessful, and studies investigating the immune microenvironment of the disease and the impact of current therapies are limited. Preclinical models that recapitulate both the disease and immune environment are essential for understanding immune-tumor interactions and to aid the identification of new and effective immunotherapies. Using an immune-competent mouse model of aggressive Myc-driven medulloblastoma, we characterized the brain immune microenvironment and changes induced in response to craniospinal irradiation, or the medulloblastoma chemotherapies cyclophosphamide or gemcitabine. The role of adaptive immunity in disease progression and treatment response was delineated by comparing survival outcomes in wildtype C57Bl/6J and in mice deficient in Rag1 that lack mature T and B cells. We found medulloblastomas in wildtype and Rag1-deficient mice grew equally fast, and that craniospinal irradiation and chemotherapies extended survival equally in wildtype and Rag1-deficient mice, suggesting that tumor growth and treatment response is independent of T and B cells. Medulloblastomas were myeloid dominant, and in wildtype mice, craniospinal irradiation and cyclophosphamide depleted T and B cells in the brain. Gemcitabine treatment was found to minimally alter the immune populations in the brain, resulting only in a depletion of neutrophils. Intratumorally, we observed an abundance of Iba1+ macrophages, and we show that CD45high cells comprise the majority of immune cells within these medulloblastomas but found that existing markers are insufficient to clearly delineate resident microglia from infiltrating macrophages. Ultimately, brain resident and peripheral macrophages dominate the brain and tumor microenvironment and are not depleted by standard-of-care medulloblastoma therapies. These populations therefore present a favorable target for immunotherapy in combination with front-line treatments

A novel technique of serial biopsy in mouse brain tumour models

<div><p>Biopsy is often used to investigate brain tumour-specific abnormalities so that treatments can be appropriately tailored. Dacomitinib (PF-00299804) is a tyrosine kinase inhibitor (TKI), which is predicted to only be effective in cancers where the targets of this drug (EGFR, ERBB2, ERBB4) are abnormally active. Here we describe a method by which serial biopsy can be used to validate response to dacomitinib treatment <i>in vivo</i> using a mouse glioblastoma model. In order to determine the feasibility of conducting serial brain biopsies in mouse models with minimal morbidity, and if successful, investigate whether this can facilitate evaluation of chemotherapeutic response, an orthotopic model of glioblastoma was used. Immunodeficient mice received cortical implants of the human glioblastoma cell line, U87MG, modified to express the constitutively-active EGFR mutant, EGFRvIII, GFP and luciferase. Tumour growth was monitored using bioluminescence imaging. Upon attainment of a moderate tumour size, free-hand biopsy was performed on a subgroup of animals. Animal monitoring using a neurological severity score (NSS) showed that all mice survived the procedure with minimal perioperative morbidity and recovered to similar levels as controls over a period of five days. The technique was used to evaluate dacomitinib-mediated inhibition of EGFRvIII two hours after drug administration. We show that serial tissue samples can be obtained, that the samples retain histological features of the tumour, and are of sufficient quality to determine response to treatment. This approach represents a significant advance in murine brain surgery that may be applicable to other brain tumour models. Importantly, the methodology has the potential to accelerate the preclinical <i>in vivo</i> drug screening process.</p></div

Histological integrity is maintained in biopsy samples.

<p>A representative image of a paraffin section from a biopsy sample. Immunohistochemical staining for phosphorylated EGFR (brown) illustrates a clear demarcation between the EGFRvIII expressing tumour cells and normal brain. Sections were counterstained with haematoxylin (blue). Scale bar represents 100μm.</p

Biopsy-related neurological morbidity is minimal and reversible.

<p>Mean neurological severity score (NSS, error bars indicate standard deviation) was assessed for all animals in control (tumour-bearing, no biopsy) and biopsy groups. Controls (n = 8) were assessed for five days. “Biopsy 1” animals (n = 22) were assessed for 5 days after one biopsy. “Biopsy 2” mice (n = 14) having recovered after biopsy one, were biopsied a second time and assessed for another 5 days. The biopsy procedure was associated with minimal morbidity and mice recovered to scores equal to controls by day 5 (NSS = 0.73 vs 0.57, p = 0.68). After a second biopsy there was minimal initial neurological injury (NSS = 1.5) on day 1 after biopsy, which returned to baseline level by day 5 (NSS = 0.81 vs 0.57, p = 0.51).</p

Micro-dissection can successfully remove a tumour biopsy sample from the murine cerebral cortex.

<p>Intraoperative images showing a fluorescent tumour <i>in situ</i> expressing GFP (A) and after removal, where the biopsied tissue is marked with a white arrow (B and C).</p

Tumour response to kinase inhibitor therapy can be observed in biopsied tissue.

<p>Tumour-bearing animals underwent surgical biopsy (pre-treatment) followed by a period of recovery before each was treated with 30mg/kg dacomitinib to inhibit EGFR. After 2 hours, a second biopsy was performed (post-treatment). Immunohistochemical detection of phosphorylated EGFR (Y1068, brown) in cryosections of biopsied tumours demonstrates that EGFR phosphorylation was significantly reduced after treatment with dacomitinib (lower row).Four representative mice are shown (#735, #736, #765 and #768). Sections were counterstained with haematoxylin (blue). Scale bar represents 100μm.</p

Characterising nitric oxide-mediated metabolic benefits of low-dose ultraviolet radiation in the mouse: a focus on brown adipose tissue

AIMS/HYPOTHESIS: Exposure to sunlight has the potential to suppress metabolic dysfunction and obesity. We previously demonstrated that regular exposure to low-doses of ultraviolet radiation (UVR) reduced weight gain and signs of diabetes in male mice fed a high-fat diet, in part via release of nitric oxide from skin. Here, we explore further mechanistic pathways through which low-dose UVR exerts these beneficial effects.METHODS: We fed mice with a luciferase-tagged Ucp1 gene (which encodes uncoupling protein-1 [UCP-1]), referred to here as the Ucp1 luciferase transgenic mouse ('Thermomouse') a high-fat diet and examined the effects of repeated exposure to low-dose UVR on weight gain and development of metabolic dysfunction as well as UCP-1-dependent thermogenesis in interscapular brown adipose tissue (iBAT).RESULTS: Repeated exposure to low-dose UVR suppressed the development of glucose intolerance and hepatic lipid accumulation via dermal release of nitric oxide while also reducing circulating IL-6 (compared with mice fed a high-fat diet only). Dietary nitrate supplementation did not mimic the effects of low-dose UVR. A single low dose of UVR increased UCP-1 expression (by more than twofold) in iBAT of mice fed a low-fat diet, 24 h after exposure. However, in mice fed a high-fat diet, there was no effect of UVR on UCP-1 expression in iBAT (compared with mock-treated mice) when measured at regular intervals over 12 weeks. More extensive circadian studies did not identify any substantial shifts in UCP-1 expression in mice exposed to low-dose UVR, although skin temperature at the interscapular site was reduced in UVR-exposed mice. The appearance of cells with a white adipocyte phenotype ('whitening') in iBAT induced by consuming the high-fat diet was suppressed by exposure to low-dose UVR in a nitric oxide-dependent fashion. Significant shifts in the expression of important core gene regulators of BAT function (Dio2, increased more than twofold), fatty acid transport (increased Fatp2 [also known as Slc27a2]), lipolysis (decreased Atgl [also known as Pnpla2]), lipogenesis (decreased Fasn) and inflammation (decreased Tnf), and proportions of macrophages (increased twofold) were observed in iBAT of mice exposed to low-dose UVR. These effects were independent of nitric oxide released from skin.CONCLUSIONS/INTERPRETATION: Our results suggest that non-burning (low-dose) UVR suppresses the BAT 'whitening', steatotic and pro-diabetic effects of consuming a high-fat diet through skin release of nitric oxide, with some metabolic and immune pathways in iBAT regulated by UVR independently of nitric oxide.</p