Abstract 4943: Visualization of endogenous melanoma initiation and progression using intravital microscopy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The death rates for malignant metastatic melanoma continue to rise because melanoma is largely refractory to existing therapies. Loss or inactivation of the tumor suppressor PTEN (Phosphatase and Tensin Homolog on Chromosome Ten) is observed in 40-50% of melanoma, and BRAF and PTEN mutations exist coincidentally in approximately 20% of melanoma cases. In mouse GEMM models, these mutations cooperate to promote metastatic melanomagenesis. However, existing methods to activate these lesions with Tyr-CreERT2 and topical tamoxifen application yields multifocal primary tumors whose origins cannot be accurately determined. To improve this model, we combined a tdTomatoLSL allele that serves as a visual marker of Cre recombination with Tyr-CreERT2;BrafCA;Ptenlox/lox to produce an animal where we can reproducibly initiate tumorigenesis in a spatiotemporally controllable manner by transiently applying extremely low doses (<20 nM) of tamoxifen to the dermis of the ear. Due to melanocyte-specific expression of the fluorescent protein tdTomato, we can track tumor progression from endogenous cells using two-photon intravital imaging. Starting from a single primary tumor on the ear, we can detect distant metastases in cervical lymph nodes and lungs via macroscopic fluorescent imaging, qRT-PCR and histology. We are using this model in combination with targeted therapy to investigate the effects of selective molecular inhibitors on the growth and metastasis of endogenous BRAFV600E/PTEN-/- metastatic melanoma at the cellular level. Preliminary studies using intravital imaging and a selective MEK1/2 inhibitor, GSK212 (Trametinib, FDA approved treatment for metastatic melanoma) show that targeted therapy causes heterogeneous reorganization of the tumor cells as early as 3 days post-treatment. After several weeks of continuous treatment, the remaining, resistant tumor cells are highly correlated spatially with bundled collagen structures detected by second harmonic generation signal, suggesting that cellular milieu strongly influences drug response. In addition, GSK212 treated animals still had lung macro-metastatic tumors, indicating that despite continuous MEK1/2 inhibition, metastatic tumor growth in the lung was still possible. In summary, we have developed an inducible endogenous model of melanoma metastasis that can be used to directly investigate both heterogeneous effects of targeted therapy at the cellular level and the efficacy of targeted therapy against metastasis. Citation Format: James E. Bear, Hailey E. Brighton, Norman E. Sharpless, David B. Darr, Kelly S. Clark. Visualization of endogenous melanoma initiation and progression using intravital microscopy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4943. doi:10.1158/1538-7445.AM2014-4943