Purpose

Approximately 95% of anticancer drugs fail in Phase 1/Phase 2 clinical trials after demonstrating sufficient efficacy in preclinical trials. One possible contributing factor is the prevalence of non-orthotopic tumor allograft or patient-derived xenograft animal models and the use of invasive or qualitative measurement tools in preclinical study of novel anticancer therapeutics. We have developed a high-throughput, manganese enhanced MRI (MEMRI) - based pipeline to test such drugs in sporadic mouse models of medulloblastoma¹. We have applied this pipeline to test the efficacy of an experimental CSF1R-inhibiting drug designed to reduce the number of tumor associated microglia and macrophages (TAMs).

Methods

Mice expressing an oncogenic form of Smoothened (SmoM2) in rare cerebellar granule precursors were made by generating mice carrying an Atoh1-CreER ² transgene and R26^{loxPSTOPloxP-SmoM2} (R26^LSL-SmoM2)³ allele as a model for the Sonic Hedgehog (SHH) subgroup of human medulloblastoma. Expression of the SmoM2 oncogene was induced by subcutaneous injection of low doses (1µg/g or 5µg/g) of Tamoxifen (TMX) at postnatal day (P)2. Pre-treatment MEMRI sessions using 7-Tesla MRI (Bruker) were conducted at postnatal week 5 (W5) and W7. MnCl₂ (50-60 mg/kg) was injected intraperitoneally 24 hours before imaging. Scan protocol: 1 min low-resolution pilot, 21min 150µm isotropic resolution T1-weighted gradient echo (GE) sequence (TE/TR = 4/30 ms; FA = 20°; FOV = 19.2 mm × 19.2 mm × 12 mm; Matrix = 128 × 128 × 80). Tumor volumes were quantified after each imaging time point by semi-automated segmentation in Amira. An absolute volume threshold of 7mm3 was applied to W5 and W7 tumor volume data to predict progression outcome (e.g. ‘progressing’ or ‘regressing’) based on preliminary data. ‘Regressing‘ animals were excluded from the study. Mice with ‘Progressing’ tumors were randomly assigned to treatment (DRUG, n=16 mice) or control (CTL, n=16 mice) arms, and fed either drug-laden or vehicle-laden chow. Imaging continued biweekly from W9 to W13. Animals were sacrificed post-imaging at W13 for either FACS or histopathology. Assessment of tumor volume changes pre/post treatment was used as the primary metric for drug efficacy.

Results

The MEMRI-based pipeline provided a sensitive method for estimating changes in tumor volume (Fig. 1). Absolute volume thresholding at 7mm³ correctly selected for ‘progressing’ tumors in 75% (n = 6/8) of mice, and for ‘regressing’ tumors in approximately 67% (n = 4/6) of mice. Response to treatment was heterogeneous, and TMX dose-dependent (Fig. 2). In both the 1µg/g and 5µg/g TMX models, immunohistological analysis showed that the number of TAMs was reduced in the DRUG compared to the CTL groups (Fig. 2). Normalized tumor volumes did not significantly differ between DRUG (Fig. 2A, red, n = 14) and CTL (Fig. 2A, green, n = 14) groups in the 1µg/g TMX model (Fig. 2C, p = 0.2582), and approximately 57% (n = 8/14) of DRUG tumors regressed after treatment, compared to 42% (n = 6/14) of CTL tumors. Heterogenous response was also observed in the 5µg/g TMX model, although normalized tumor volumes were statistically different between DRUG (n = 12) and CTL (n = 12) groups in the 5µg/g TMX model (Fig. 2C, p = 0.043). Approximately 83% of DRUG tumors (Fig. 2B, red, n = 10/12) regressed after treatment, compared to 33% (Fig. 2B, green, n = 4/12) of CTL tumors. These preliminary results have guided ongoing study of drug efficacy in the 5µg/g TMX model to improve the statistical power and verify whether there is a real response to the TAM-targeted drug.

Discussion and Future Directions

Our high-throughput, MEMRI-driven preclinical pipeline revealed unanticipated heterogeneity between individual tumors in terms of growth rates, and in the progression of tumors within an individual animal. This highlights the critical need for in vivo imaging in preclinical studies of anticancer drugs. In future, we plan to explore targeted imaging methods, such as immune cell molecular imaging, to extend and improve our analysis of TAM-targeted therapy in medulloblastoma.

Acknowledgements

This work was made possible by NIH grants R01NS038461 (DHT) and R01CA192176 (ALJ).