Optimization, Characterization, and Comparison of Two Luciferase-Expressing Mouse Glioblastoma Models.

Glioblastoma (GBM) is the most aggressive brain cancer. To model GBM in research, orthotopic brain tumor models, including syngeneic models like GL261 and genetically engineered mouse models like TRP, are used. In longitudinal studies, tumor growth and the treatment response are typically tracked with in vivo imaging, including bioluminescence imaging (BLI), which is quick, cost-effective, and easily quantifiable. However, BLI requires luciferase-tagged cells, and recent studies indicate that the luciferase gene can elicit an immune response, leading to tumor rejection and experimental variation. We sought to optimize the engraftment of two luciferase-expressing GBM models, GL261 Red-FLuc and TRP-mCherry-FLuc, showing differences in tumor take, with GL261 Red-FLuc cells requiring immunocompromised mice for 100% engraftment. Immunohistochemistry and MRI revealed distinct tumor characteristics: GL261 Red-FLuc tumors were well-demarcated with densely packed cells, high mitotic activity, and vascularization. In contrast, TRP-mCherry-FLuc tumors were large, invasive, and necrotic, with perivascular invasion. Quantifying the tumor volume using the HALO® AI analysis platform yielded results comparable to manual measurements, providing a standardized and efficient approach for the reliable, high-throughput analysis of luciferase-expressing tumors. Our study highlights the importance of considering tumor engraftment when using luciferase-expressing GBM models, providing insights for preclinical research design.

Characterization and comparison of human glioblastoma models.

Glioblastoma (GBM) is one of the deadliest cancers. Treatment options are limited, and median patient survival is only several months. Translation of new therapies is hindered by a lack of GBM models that fully recapitulate disease heterogeneity. Here, we characterize two human GBM models (U87-luc2, U251-RedFLuc). In vitro, both cell lines express similar levels of luciferase and show comparable sensitivity to temozolomide and lapatinib exposure. In vivo, however, the two GBM models recapitulate different aspects of the disease. U87-luc2 cells quickly grow into large, well-demarcated tumors; U251-RedFLuc cells form small, highly invasive tumors. Using a new method to assess GBM invasiveness based on detecting tumor-specific anti-luciferase staining in brain slices, we found that U251-RedFLuc cells are more invasive than U87-luc2 cells. Lastly, we determined expression levels of ABC transporters in both models. Our findings indicate that U87-luc2 and U251-RedFLuc GBM models recapitulate different aspects of GBM heterogeneity that need to be considered in preclinical research.

Flow Rate and Apparent Volume of Cerebrospinal Fluid in Rhesus Macaques (Macaca mulatta) Based on the Pharmacokinetics of Intrathecally Administered Inulin.

Cerebrospinal fluid (CSF) flow rate and volume are fundamental to the design and interpretation of preclinical pharmacokinetics and pharmacodynamics studies in NHP. To determine the values of CSF flow rate and volume, we evaluated the plasma and CSF pharmacokinetics of inulin, an inert polysaccharide tracer, in 5 rhesus macaques with CSF ventricular res- ervoirs and lumbar ports; these reservoirs and ports facilitate humane intrathecal administration and serial CSF sampling in unanesthetized macaques. Inulin was administered intrathecally via the CSF ventricular reservoir (n = 3), followed by the collection of lumbar CSF via the lumbar port and plasma. The contribution of dietary inulin was evaluated by using pre- and postprandial inulin plasma concentrations (n = 2) and a feed analysis of the NHP diet. Inulin concentrations were quantified using ELISA. Pharmacokinetic parameters were calculated by using noncompartmental methods. Daily diet was analyzed for inulin by using Official Method no. 997.08 of AOAC International. In male rhesus macaques, the mean CSF flow rate, established via inulin clearance after IT administration, was 0.018 ± 0.003 mL/min; mean CSF volume, established based on apparent volume of distribution, was 10.17 ± 0.63 mL. In plasma, inulin was quantifiable in all pre-administration samples and increased over the sampling period, precluding interpretation of plasma pharmacokinetics. Evaluation of the effect of diet on plasma concentrations established quantifiable inulin levels that showed minimal variation relative to the prandial state. Analysis of the feed detected 5 inulin types ranging from 1100 to 1440 mg per100 g. The diet was the source of detectable pre-administration inulin plasma concentrations, whereas inulin was not detected in CSF before inulin administration.

A phase I trial of lenalidomide and radiotherapy in children with diffuse intrinsic pontine gliomas or high-grade gliomas.

PURPOSE: This study was performed to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D) of the immunomodulatory agent, lenalidomide, when administered daily during 6 weeks of radiation therapy to children with newly diagnosed diffuse intrinsic pontine glioma (DIPG) or high-grade glioma (HGG) PATIENTS & METHODS: Children and young adults < 22 years of age with newly diagnosed disease and no prior chemotherapy or radiation therapy were eligible. Children with HGG were required to have an inoperable or incompletely resected tumor. Eligible patients received standard radiation therapy to a prescription dose of 54-59.4 Gy, with concurrent administration of lenalidomide daily during radiation therapy in a standard 3 + 3 Phase I dose escalation design. Following completion of radiation therapy, patients had a 2-week break followed by maintenance lenalidomide at 116 mg/m2/day × 21 days of a 28-day cycle. RESULTS: Twenty-nine patients (age range 4-19 years) were enrolled; 24 were evaluable for dose finding (DIPG, n = 13; HGG, n = 11). The MTD was not reached at doses of lenalidomide up to 116 mg/m2/day. Exceptional responses were noted in DIPG and malignant glioma (gliomatosis cerebri) notably at higher dose levels and at higher steady state plasma concentrations. The primary toxicity was myelosuppression. CONCLUSION: The RP2D of lenalidomide administered daily during radiation therapy is 116 mg/m2/day. Children with malignant gliomas tolerate much higher doses of lenalidomide during radiation therapy compared to adults. This finding is critical as activity was observed primarily at higher dose levels suggesting a dose response.

Plasma and cerebrospinal fluid pharmacokinetics of the DNA methyltransferase inhibitor, 5-azacytidine, alone and with inulin, in nonhuman primate models.

BACKGROUND: Epigenetic modifiers are being investigated for a number of CNS malignancies as tumor-associated mutations such as isocitrate dehydrogenase mutations (IDH1/IDH2) and H3K27M mutations, which result in aberrant signaling, are identified. We evaluated the CNS exposure of the DNA methyltransferase inhibitor, 5-azacytidine (5-AZA), in preclinical nonhuman primate (NHP) models to inform its clinical development for CNS tumors. METHODS: 5-AZA and 5-AZA+Inulin pharmacokinetics (PK) were evaluated in NHPs (n = 10) following systemic (intravenous [IV]) and intrathecal (intraventricular [IT-V], intralumbar [IT-L], and cisternal [IT-C]) administration. Plasma, cerebrospinal fluid (CSF), cortical extracellular fluid (ECF), and tissues were collected. 5-AZA levels were quantified via ultra-high-performance liquid chromatography with tandem mass spectrometric detection assay and inulin via ELISA. PK parameters were calculated using noncompartmental methods. RESULTS: After IV administration, minimal plasma exposure (area under the curve [AUC] range: 2.4-3.2 h*µM) and negligible CSF exposure were noted. CSF exposure was notably higher after IT-V administration (AUCINF 1234.6-5368.4 h*µM) compared to IT-L administration (AUCINF 7.5-19.3 h*µM). CSF clearance after IT administration exceeded the mean inulin CSF flow rate of 0.018 ± 0.003 ml/min as determined by inulin IT-V administration. 5-AZA IT-V administration with inulin increased the 5-AZA CSF duration of exposure by 2.2-fold. IT-C administration yielded no quantifiable 5-AZA ECF concentrations but resulted in quantifiable tissue levels. CONCLUSIONS: IT administration of 5-AZA is necessary to achieve adequate CNS exposure. IT administration results in pronounced and prolonged 5-AZA CSF exposure above the reported IC50 range for IDH-mutated glioma cell lines. Inulin administered with 5-AZA increased the duration of exposure for 5-AZA.

Autologous Fat Grafting Does Not Increase Risk of Oncologic Recurrence in the Reconstructed Breast.

INTRODUCTION: Autologous fat grafting (AFG) is a popular and effective method of breast reconstruction after mastectomy; however, the oncological safety of AFG remains in question. The aim of this study was to determine whether AFG increases the risk of cancer recurrence in the reconstructed breast. METHODS: A matched, case-control study was conducted from 2000 to 2017 at the senior author's institution. Inclusion was limited to female patients who underwent mastectomy and breast reconstruction with or without AFG. Data were further subdivided at the breast level. χ analyses were used to test the association between AFG status and oncologic recurrence. A Cox proportional-hazards model was constructed to assess for possible differences in time to oncologic recurrence. The probability of recurrence was determined by Kaplan-Meier analyses and confirmed with log-rank testing. RESULTS: Overall, 428 breasts met study criteria. Of those, 116 breasts (27.1%) received AFG, whereas 312 (72.9%) did not. No differences in the rates of oncologic recurrence were found between the groups (8.2% vs 9.0%, P < 1.000). Unadjusted (hazard ratio = 1.03, confidence interval = 0.41-2.60, P < 0.957) and adjusted hazard models showed no statistically significant increase in time to oncologic recurrence when comparing AFG to non-AFG. In addition, no statistical differences in disease-free survival were found (P = 0.96 by log rank test). CONCLUSION: Autologous fat grafting for breast reconstruction is oncologically safe and does not increase the likelihood of oncologic recurrence. Larger studies (eg, meta analyses) with longer follow-up are needed to further elucidate the long-term safety of AFG as a reconstructive adjunct.

Characterizing the pharmacokinetics of panobinostat in a non-human primate model for the treatment of diffuse intrinsic pontine glioma.

PURPOSE: Diffuse intrinsic pontine glioma (DIPG) is one of the deadliest forms of childhood cancers. To date, no effective treatment options have been developed. Recent drug screening studies identified the HDAC inhibitor panobinostat as an active agent against DIPG cells lines and animal models. To guide in the clinical development of panobinostat, we evaluated the CNS pharmacokinetics of panobinostat using CSF as a surrogate to CNS tissue penetration in a pre-clinical nonhuman primate (NHP) model after oral administration. METHODS: Panobinostat was administered orally to NHP (n = 3) at doses 1.0, 1.8, 2.4, and 3.0 mg/kg (human equivalent dose: 20, 36, 48, 60 mg/m2, respectively). The subjects served as their own controls where possible. Serial, paired CSF and plasma samples were collected for 0-48 h. Panobinostat was quantified via a validated uHPLC-MS/MS method. Pharmacokinetic (PK) parameters were calculated using non-compartmental methods. RESULTS: CSF penetration of panobinostat after systemic delivery was low, with levels detectable in only two subjects. CONCLUSION: The CSF penetration of panobinostat was low following oral administration in this pre-clinical NHP model predictive of human PK.