Continuous and bolus intraventricular topotecan prolong survival in a mouse model of leptomeningeal medulloblastoma

Abstract

Leptomeningeal metastasis remains a difficult clinical challenge. Some success has been achieved by direct administration of therapeutics into the cerebrospinal fluid (CSF) circumventing limitations imposed by the blood brain barrier. Here we investigated continuous infusion versus bolus injection of therapy into the CSF in a preclinical model of human Group 3 medulloblastoma, the molecular subgroup with the highest incidence of leptomeningeal disease. Initial tests of selected Group 3 human medulloblastoma cell lines in culture showed that D283 Med and D425 Med were resistant to cytosine arabinoside and methotrexate. D283 Med cells were also resistant to topotecan, whereas 1 μM topotecan killed over 99% of D425 Med cells. We therefore introduced D425 Med cells, modified to express firefly luciferase, into the CSF of immunodeficient mice. Mice were then treated with topotecan or saline in five groups: continuous intraventricular (IVT) topotecan via osmotic pump (5.28 μg/day), daily bolus IVT topotecan injections with a similar daily dose (6 μg/day), systemic intraperitoneal injections of a higher daily dose of topotecan (15 μg/day), daily IVT pumped saline and daily intraperitoneal injections of saline. Bioluminescence analyses revealed that both IVT topotecan treatments effectively slowed leptomeningeal tumor growth in the brains. Histological analysis showed that they were associated with localized brain necrosis, possibly due to backtracking of topotecan around the catheter. In the spines, bolus IVT topotecan showed a trend towards slower tumor growth compared to continuous (pump) IVT topotecan, as measured by bioluminescence. Both continuous and bolus topotecan IVT showed longer survival compared to other groups. Thus, both direct IVT topotecan CSF delivery methods produced better anti-medulloblastoma effect compared to systemic therapy at the dosages used here.

Fig 1. D425MED cells, but not D283MED, are sensitive to topotecan in culture.

(A-B) D283 and clone 5 of D425 medulloblastoma cells, expressing firefly luciferase, were seeded at 2×10³ cells/well into 96-well plates, and methotrexate (MTX), cytosine arabinoside (ARA-C) or topotecan (TPT) were added for 72 h. Cells were analyzed for residual bioluminescence (Radiance) as a measurement of cells surviving following treatment. Data are averages of duplicate measurements of duplicate wells ±SEM. 72 h IC50 for TPT was 248 μM in D283 cells (A) and 35 nM in D425 cells (B). IC50 was not reached for MTX or ARA-C in either cell line. (C) D425 clones #4, #5 and #7 expressing firefly luciferase were seeded at 1×10⁴ cells/well into a 96-well plate and exposed to the indicated concentrations of topotecan for 96 h between days 2 and 6 after plating, with drug/medium replaced on day 2 and on day 4 after plating. Bioluminescence was assessed 6 days after plating. Data are mean measurements of quadruplicate wells ±SEM. The 96 h IC50s were 2.9 nM (clone #7), 1.8 nM (clone #5) and 3.6 nM (clone #4), with mean IC50 of 2.8 nM ± 0.52 (SEM). Error bars for most data points are smaller than the symbols and are not visible.

Fig 2. IVT topotecan slows leptomeningeal growth of D425 medulloblastoma cells in nude mice.

D425-ff-luc cells were inoculated into the cisterna magna of nude mice. The following day treatment with topotecan was started via the indicated route. Bioluminescence was evaluated until mice showed clinically apparent signs of tumor and were euthanized. (A) Bioluminescence imaging at day 14, which was the last imaging session when all mice in all groups were still alive. (B) Mean ± SEM of bioluminescence of each group. Means represent evaluations when all mice in the group were still alive, after which the curve is no longer shown. Below are p-values (log rank) comparing bioluminescence between the groups on day 14, which was the last imaging session when all mice were still alive. Saline IP, n = 8 mice; Saline IVT pump, n = 4; TPT IP, n = 9; TPT IVT pump, n = 6; TPT IVT bolus, n = 7.

Fig 3. IVT topotecan prolongs survival of mice with leptomeningeal D425 medulloblastoma compared to bolus IP topotecan.

Kaplan-Meier survival curves of mouse groups are shown. (A) comparison of all groups. (B) Daily IP topotecan versus daily IP saline control. (C) Daily IP topotecan, daily IVT topotecan, or continuous IVT topotecan infusion via pump. Median survival (marked by asterisks) and number of mice in each group are listed in the key next to panel A. Comparison between pairs of Kaplan-Meier curves, calculated by log rank (Mantel-Cox) in Prism, are below panel C.

Fig 4. IVT topotecan preferentially slows leptomeningeal tumor growth in brains versus spines.

Bioluminescence of brains and spines calculated at each time point up to the first death in each group. Shown: mean ± SEM for each group. (A) Ratios of spine-to-brain radiance measurements illustrate the relatively-faster increase in spine radiance compared to brain radiance in IVT TPT groups compared to the non-IVT groups. (B) Brain radiance measurements reveal more effective suppression of tumor growth in brains of TPT IP mice compared to saline IP in brains of TPT IVT (bolus or pump) mice compared to TPT IP and in brains of TPT IVT bolus mice compared to TPT IVT pump. (C) Spine radiance measurements reveal more effective tumor growth suppression in spines of TPT IVT bolus mice compared to TPT IP mice. There was a trend toward significance in spines of mice treated with TPT IVT bolus compared to TPT IVT pump, but it did not reach significance levels. p-values were calculated for each two groups using unpaired t-tests at each of the time points.

Fig 5. Leptomeningeal spread of D425 medulloblastoma cells is extensive.

H&E stain of cerebellum (A; sagittal section) and spine (B-D; cross-sections) from a control mouse (IVT saline pump) euthanized at the time of tumor symptoms. Sections show extensive leptomeningeal spread of tumor cells (large black arrowheads) around the brain and the spinal cord. Asterisks mark some of the nerve roots entrapped within the leptomeningeal medulloblastoma.

Fig 6. IVT topotecan delivered via intracranial cannula was associated with brain necrosis.

Representative H&E-stained sections of brains from mice that received either IVT saline by continuous infusion (A-B; no necrosis) or IVT topotecan by continuous infusion (C-D; extensive necrosis). Brains of other assessible mice that received IVT topotecan by bolus or by pump also showed necrosis in the region of the hippocampus and region superior to it. Mice that received IVT continuous saline or IP topotecan or IP saline did not show such necrosis. Asterisk in panel A marks normal hippocampus and asterisk in panel C marks region of extensively-damaged hippocampus.