A Hematogenous Route for Medulloblastoma Leptomeningeal Metastases

Abstract

While the preponderance of morbidity and mortality in medulloblastoma patients are due to metastatic disease, most research focuses on the primary tumor due to a dearth of metastatic tissue samples and model systems. Medulloblastoma metastases are found almost exclusively on the leptomeningeal surface of the brain and spinal cord; dissemination is therefore thought to occur through shedding of primary tumor cells into the cerebrospinal fluid followed by distal re-implantation on the leptomeninges. We present evidence for medulloblastoma circulating tumor cells (CTCs) in therapy-naive patients and demonstrate in vivo, through flank xenografting and parabiosis, that medulloblastoma CTCs can spread through the blood to the leptomeningeal space to form leptomeningeal metastases. Medulloblastoma leptomeningeal metastases express high levels of the chemokine CCL2, and expression of CCL2 in medulloblastoma in vivo is sufficient to drive leptomeningeal dissemination. Hematogenous dissemination of medulloblastoma offers a new opportunity to diagnose and treat lethal disseminated medulloblastoma.

Keywords: brain tumors; circulating tumor cells; medulloblastoma; metastases; pediatric cancer.

Figure 1:. Circulating Medulloblastoma Tumor Cells in Therapy-Naïve Humans

a) Read counts from whole genome sequencing of simultaneously collected, patient matched primary tumor, metastatic tumor, and blood in one representative metastatic patient identifies a somatic mutation (C-T) found to be heterozygous in the metastasis (VAF 47.5%), subclonal in the primary tumor (12.5%), and extremely subclonal (1.3%) in the patient matched blood, suggesting the presence of circulating tumor cells. b) Clonality comparison of somatic mutations identified by whole genome sequencing of peripheral blood as compared to primary tumor versus patient matched metastases reveals that putative circulating tumor cells may carry mutations restricted to the metastatic compartment and mutations shared between the primary tumor and the metastasis. See also Figure S1 and Table S1. c) ImageStream analysis shows NCAM+ve/CD45-ve cells in the blood of MB patients sampled at diagnosis suggestive of CTC cells. d) Representative high power images of MB CTC in one patient display NCAM positivity and abnormal morphology. e) the same patient also shows diffuse NCAM positivity in MB (spinal metastasis pre-treatment), scale bars 50μm left, 10μm right panel. See also Figure S1.

Figure 2:. Circulating Medulloblastoma Cells Generate Leptomeningeal Medulloblastoma and Vice Versa

a) Peripheral blood from a murine GEMM model of metastatic MB, as well as immune deficient mice grafted with three patient derived xenograft models of human metastatic medulloblastoma analyzed by flow cytometry demonstrates GFP^+ve cells in the blood consistent with the presence of rare circulating medulloblastoma cells (1 in 1000 to 1 in 100000). Prevalence of CTC in mice injected with different lines is summarized in inlet table. (Scale bars, 1mm) b) Primary human PDX medulloblastoma cells or matched spinal metastasis were serially isolated by FACS and grafted into the cerebellum of NSG mice, (100 cells each) followed by observation and autopsy at clinical end-point. c) There is no significant difference in survival between metastatic derived grafts (n=22) and primary tumor derived grafts (n=17) (Log Rank test p=0.719). d) Mice grafted in the cerebellum with metastatic derived cells (n=15) have a greatly increased burden of spinal leptomeningeal metastases at endpoint as compared to mice grafted with primary tumors (n=7), as assessed by flow-cytometry. (Mann-Whitney U test p=0.040) In the box plots center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles e) Xenografting of patient derived medulloblastoma xenografts, or allografting of GEMM murine medulloblastomas into the flank of NSG mice was followed by survival surgery to remove the flank mass at a predetermined size, followed by post-operative care and monitoring reveals leptomeningeal metastases (scale bars, 200μm). f) Some, but not all mice develop leptomeningeal metastases in a delayed fashion after removal of their flank medulloblastoma graft (*ONS76 flank implants were sub-totally resected).

Figure 3:. Hematogenous Dissemination of Medulloblastoma Between Parabiont Mice

a) Schematic of the workflow to assess hematogenous dissemination of medulloblastoma using parabiont NSG twins. b) Evans blue administered by tail vein injection under general anesthesia demonstrates patency of the vascular anastomosis in the parabionts. c) Primary medulloblastoma is observed in the donor twin sacrificed after twin separation at the time of symptoms. The recipient is observed until endpoint, at which time leptomeningeal metastases were observed in three out of six parabiont pairs for PDX line Med-411FH. See also Figure S3. d) PDX lines MMB and MMS also display hematogenous tropism to leptomeninges when used implanted in parabiont mice, scale bars 50μm. e) Frequency of hematogenous dissemination in MB parabiont, see also figure S3.

Figure 4:. CCL2 Overexpression in Human Leptomeningeal Metastases

a) CCL2 is differentially expressed between human primary and metastatic Group 3 medulloblastoma surgical samples as compared by expression microarray (p=0.041). See also Table S2. b) Expression of CCL2 is associated with metastatic status 2 and 3 in a cohort of 70 Grp3 MB with clinical annotation, 46.6% of M2/3 express CCL2 vs 20% in the M0/1 group. c) Human CCL2 resides on chromosome 17q, a region frequently gained in Group 3. Gain of chromosome 17q is significantly more common in patients with leptomeningeal dissemination for both Group 3 and Group 4 cohorts. In contrast, CCL2 expression is only marginally correlated with M2/3 in Shh patients, and the trend (23.5% CCL2 positive M1/0 vs 43.5% CLL2 positive) does not reach statistical significance (p=0.2462 Fisher’s exact test). d) 10q and 17q status as determined by 450K methylation array and 250K array (ONS76*)

Figure 5:. The CCL2/CCR2 Axis drives Medulloblastoma Leptomeningeal Dissemination.

a) The ONS76 line was infected with CCL2 alone or in combination with CCR2 expressing or control lentiviruses. Control ONS76 xenografts are poorly metastatic (5/10 animals) when xenografted into NSG mice, where-as expression of either CCL2 (9/10 animals) or CCR2 (5/5 animals), or both CCL2 and CCR2 (5/5 animals) are able to increase leptomeningeal dissemination (left side photomicrographs, scale bars H&E 50μm, fluorescence 2mm). Metastasis driven by CCL2 or CCR2 overexpression are significantly larger than the metastasis found on the spinal cords of ONS76-CTL xenografted mice (right side box-plot, p=0.023 and p=0.037 respectively, Mann-Withney U-test). In the box plots center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles b) Similarly, overexpression of CCL2 the poorly metastatic medulloblastoma patient derived line MB002 significantly increases prevalence of metastasis (18% to 61%, p=0.029, Fisher’s exact test with mid P adjustment α=0.05, left side photmicrographs, scale bars H&E 50μm, fluorescence 2mm) without affecting the size of the metastatic lesions (right side box-plot). In the box plots center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles c) Knockdown of CCL2 by lentiviral mediated shRNAs (single hairpin and combination of two hairpins) in highly metastatic human D425S medulloblastoma cells show decreased protein expression of CCL2 by Western blotting and diminished metastatic deposits after in vivo intracranial implantation in NSG mice (scale bars, 1mm). d) Summary Table of the statistical significance of all comparisons (Fisher’s exact test with mid P adjustment, α=0.05). e-f) CCR2 KO in the tumor microenvironment in vivo attenuates the metastatic behavior of MB cells in both CB and flank implants. Med-411FH were used for orthotopic flank xenografts of CCR2−/−;NSG or CCR2wt;NSG littermates. The absence of CCR2 leads to a trend in size decrease of metastatic deposits in the leptomeninges in intracranial implants, which is more pronounced than in flanks (p=0.051 and p=0.314 respectively, Mann-Withney U-test). Boxes highlight mice with higher LMD. In the box plots center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles (Scale bars, 1mm (E and F).

Figure 6:. The CCL2/CCR2 Axis Drives Leptomeningeal Dissemination In Vivo

NSG mice implanted with MPLuc allografts develop spinal metastasis with higher frequency when both CCL2 and CCR2 are overexpressed (1/13 vs 7/13, p=0.006 Fisher’s Exact Test with mid-P adjustment, α=0.05), the black arrows point to BLI signals from the spinal metastasis observable 15 days after implantations of the tumor cells (See also Figure S6). b) Nestin-TVA transgenic mice were injected with viruses to transfer either Shh alone, Shh+CCL2, Shh+CCR2, Shh+CCL2+CCR2, or CCL2+CCR2 alone. Mice injected with CCL2+CCR2 did not develop medulloblastoma. All other genotypes were observed to have a similar rate of medulloblastoma free survival (Log-Rank test p=0.217). c) While Shh virus alone leads to localized medulloblastoma in vivo, the addition of CCL2 and/or CCR drives the development of leptomeningeal metastases. Expression of CCL2, or CCR2 has no significant impact on the incidence of primary tumor formation (Chi-square test p=0.107), but does have a significant and dramatic effect on the incidence of leptomeningeal metastases (Chi-square test p=0.018). d) Primary and metastatic medulloblastoma sections from Nestin-TVA mice infected with Shh and mCherry-CCL2 viruses demonstrates that CCL2 expression is subclonal in the primary tumor, but is highly clonally selected and ubiquitous in the metastases. e) Primary and metastatic medulloblastoma sections from Nestin-TVA mice infected with Shh, Shh+CCL2, and Shh+ CCL2+CCR2 viruses demonstrates that CCL2 expression - revealed by IHC for CLL2 mcherry tag - is subclonal in the primary tumor, but is clonally selected in the meningeal metastasis. Scale bars 50μm.