Longitudinal MEMRI Characterization of a Novel Mouse Medulloblastoma Model
Harikrishna Rallapalli1, Eugenia Rafaela Volkova1, I-Li Tan2, Alexandre Wojcinski2, Alexandra L Joyner2, and Daniel H Turnbull1

1Skirball Institute and Radiology, New York University School of Medicine, New York, NY, United States, 2Developmental Biology, Sloan Kettering Institute, New York, NY, United States

Synopsis

In this work, we describe a powerful longitudinal Manganese-enhanced magnetic resonance imaging (MEMRI) strategy to characterize a novel mouse medulloblastoma model. An activated Smoothened mutation was engineered to induce proliferative growth in the cerebellum. Lesions were monitored using MEMRI up to postnatal day P100, and 3D tumors were segmented for quantitative volumetric analysis. Qualitative analysis has shown a ~50% chance of regression overall (n=21), and preliminary quantitation has suggested a combined progression/regression growth model. With this model, we aim to guide diagnostic decisions from early timepoints and quantify therapeutic efficacy.

Motivation

In vivo imaging modalities provide powerful tools for the noninvasive longitudinal characterization of preclinical cancer models. Medulloblastoma (MB) is the most common malignant brain tumor in children, and the subject of intense research, much of which involves mouse models. Manganese-enhanced magnetic resonance imaging (MEMRI) produces unparalleled images of the cerebellum, the site of most MBs [1,2]. For this reason, longitudinal MEMRI of preclinical medulloblastoma models enables analysis of the region of origin, tumor progression, and treatment response evaluation. In this study, we present the initial MEMRI characterization of a novel mouse medulloblastoma model with an activating mutation in the Smo gene, which is expected to be more aggressive than previous studies in Ptch1 knockout mice [1].

Methods

SmoM2 mice were engineered by crossing Atoh1-CreER [3] male mice with homozygous R26-floxedSTOP-SmoM2 females [4]. The SmoM2 mutation was induced by subcutaneous injection of low dose (1µg/g) Tamoxifen (TMX) at postnatal day P2. Weekly imaging sessions using 7-Tesla MRI (Bruker) began at postnatal day P21. MnCl2 (50-60 mg/kg) was injected intraperitoneally 24 hours before imaging. Scan protocol: 1 min low-resolution pilot, 2hr 100µm resolution T1-weighted self-gated gradient echo (GE) sequence (P21 only) (TE/TR = 3.6/50 ms; FA = 40°; FOV = 25.6 mm × 25.6 mm × 25.6 mm; Matrix = 256 × 256 × 256), and 30 min 150µm resolution T1-weighted GE sequence (TE/TR = 4/15 ms; FA = 18°; FOV = 19.2 mm × 19.2 mm × 12 mm; Matrix = 128 × 128 × 80). Images were analyzed in 3-space using Amira and Fiji. Morphological characterization was corroborated with histology as shown in Fig 1.

Results

Longitudinal MEMRI results are illustrated in Fig 2. Based on our preliminary results, all SmoM2 mice have pre-neoplastic lesions (n=21), while approximately half develop into full tumor morphology (n=11). Of the mice with tumors, approximately 50% develop bilateral tumors and the remaining develop tumors in either the right or left hemisphere. General disease progression is as follows: at ~P21, small lesions are apparent in the majority of interlobule spaces including the mid vermis; at ~P50, regions of proliferative lesion thickening are apparent and smaller lesions regress; at ~P90 significant tumor encroachment into the forebrain as well as expansion of the third and fourth ventricles are apparent. Tumors were observed to originate in the posterior hemispheres, shift and compress the normal appearing cerebellum as they progress, and finally encroach into the forebrain. Noticeable symptoms - including delayed tail-pull reflex, ataxia, and hydrocephalus - in SmoM2 mice were apparent as early as P70.

In addition to qualitative understanding of tumor progression, we have manually segmented and quantified tumor volume at these key timepoints in an effort to produce a unified growth model. A selection (n=4) of the results from our preliminary analysis is shown in Fig 3. Estimated tumor volume doubling time is approximately 4.5 days at early timepoints (<P80). Growth plateaus have been observed at later timepoints, primarily due to insufficient free space for tumors to grow into. In future estimations, we will employ cluster analysis-based curve fitting to model both progressing and regressing tumors.

Discussion and Future Direction

We have developed a powerful longitudinal MEMRI strategy for characterization of a novel mouse medulloblastoma model. Preliminary qualitative assessment has provided a morphological understanding of disease progression. Further quantitative analysis will produce a robust growth curve model that has the potential to guide therapeutic decisions from early timepoints. Given this model, it will be possible to compare many medulloblastoma models (e.g., SmoM2 point mutation vs Ptch1 knockout) to truly understand the disease progression of the many medulloblastoma subtypes and quantitatively evaluate the efficacy of therapeutic drugs.

Acknowledgements

Acknowledgements: Research supported by NIH grants R01NS038461 and R01HL078665. Special thanks to Nathanael Kim for his assistance with image analysis.

References

1. Suero-Abreu GA, Raju GP, Aristizábal O et al,,In vivo Mn-enhanced MRI for early tumor detection and growth rate analysis in a mouse medulloblastoma model. Neoplasia 2014; 16: 993-1006.

2. Szulc KU, Lerch JP, Nieman BJ, et al. 4D MEMRI atlas of neonatal FVB/N mouse brain development. NeuroImage 2015; 118: 49-62.

3. Machold R, Fishell G. Math1 is expressed in temporally discrete pools of cerebellar rhombic-lip neural progenitors. Neuron 2005; 48: 17-24.

4. Mao J, Ligon KL, Rakhlin EY, et al. A novel somatic mouse model to survey tumorigenic potential applied to the Hedgehog pathway. Cancer Res. 2006; 66: 10171-8.

Figures

Figure 1. MEMRI results show exquisite correlation to histology. Key features are apparent in both MEMRI and histology in sagittal section. a) expansion of the fourth ventricle b) abnormal lesion proliferation in the anterior right hemisphere c) compression of lobule in right hemisphere d) significant tumor encroachment rostrally into the forebrain, up to the dentate gyrus.

Figure 2. Longitudinal disease state monitoring using MEMRI. 2D and 3D visualization of disease state at key timepoints is made possible by MEMRI.

Figure 3. Preliminary quantitative volumetric analysis suggests combined growth/regression model of disease progression. From this analysis, it is apparent a single-modal growth paradigm is not sufficient to describe both progression and regression. Our current efforts are focused on producing an accurate model to guide diagnostic decisions from early timepoints. *Mutant 3 was sacrificed on P90 due to complications associated with significant tumor burden.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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