Future Directions

Areas of interest for future investigations of this project include: histological characterization of enhancing and nonenhancing melanoma brain metastases, use of this model for testing chemotherapeutic potential or efficacy in treating brain metastases. And lastly, the use of bSSFP as an addendum clinical sequence for detection of nonenhancing metastases in the clinical setting, and using other future MRI studies for studying enhancing and nonenhancing brain metastases.

3.5.1 Histological and Immunohistochemical Characterization of Enhancing and Nonenhancing Melanoma Brain Metastases

An important area of future research for melanoma brain metastases is the immunohistochemical characterization of enhancing and nonenhancing melanoma brain metastases using angiogenic and BBB markers. By further investigating markers that are uniquely expressed and associated with enhancing and nonenhancing brain metastases, respectively, we will be able to gain a better understanding of their inherent differences. Ultimately we believe that this will help to propel better treatment planning in terms of drug development. Molecular factors that would be of interest for testing include BRAF which is involved with growth, survival, VEGFA and STAT3 which are involved with angiogenesis, and GLUT-1, which is involved with BBB development.

It would also be interesting to histologically evaluate tissue sections corresponding to contrast enhancing and nonenhancing brain metastases visualized by MRI at each time point.

This would permit us to compare tissue morphologies over time and examine whether subtle differences exist between tumour types that were not evident at the experimental endpoint.

3.5.2 Testing Therapeutic Efficacy using a Clinically Relevant Model

The goal of treatment, is to treat metastases as early as possible; however, the reality is that most brain metastases are detected at later stages of disease. The clinically relevant model we have established has the potential to be used in testing the efficacy of novel chemotherapeutic agents and in the evaluation of alternative methods for delivery of chemotherapeutic agents to target sites. Use of this model would aid tremendously in observing how melanoma brain metastases would respond to these therapies and potency of these drugs. Also there is the potential of this model to be used with other therapeutic modalities such as radiotherapy, radiosurgery, immunotherapy and targeted therapy (19,20). This model could also be used for different stages of disease onset. We are confident that this clinically relevant model of melanoma has the potential to be used in the evaluation of new treatment options.

We also aim to use this model to test the efficiency of alternative methods for drug delivery across the intact BBB. Some attractive proposed mechanisms include the use of neural stem/progenitor cells (NSPCs) loaded with 5-fluorocytosine (5-FC) pro-drug (inactive drug) of the chemotherapeutic agent and NSPCs loaded with the enzyme cytosine deaminase (CD) used to activate the pro-drug (21). Nanocarriers/nanoparticles such as poly (n-butyl cyanoacrylate) (PBCA) (22) and polyethylene glycol (PEG) liposomal nanocarriers (23) are being developed as vehicle agents for drug delivery. Focused ultrasound (FUS) guided MRI techniques have also been used as a method to temporarily disrupt the BBB (24,25). Our model could be used to test the delivery and efficacy of these new drug carrier methods.

3.5.3 Translation of the bSSFP Pulse Sequence for Brain Metastasis Detection in the Clinic The goal of establishing a clinically relevant animal model is to be able to translate preclinical findings to the clinical setting. Currently no reports in the literature have shown

bSSFP used for the detection of human melanoma metastases in visceral organs or the brain. We have shown that nonenhancing metastases are detectable on bSSFP images at early time points. This reinforces the need to use alternative clinical sequences because brain metastases that do not enhance may be missed using current brain metastasis detection methods. Also another important point to mention is that amelanotic melanoma brain metastases may also appear isointense in T1w and T2w images as well as in bSSFP. Thus, if these metastases do not enhance using Gd-DTPA then they would most certainly be missed in brain metastasis detection. The implementation of bSSFP in clinical imaging protocols would increase the potential for earlier detection of nonenhancing metastases. However, this would only apply to metastases that do not appear isointense (in the case of melanoma brain metastases).

3.5.4. Future MRI Studies for Melanoma Brain Metastases

In future MRI studies we would like to use iron oxide contrast agents to evaluate the vascularity of enhancing and nonenhancing brain metastases. As a pilot project, we have imaged two mouse brains at day 14 and day 31 pre- and post-USPIO (Molday) contrast agent. Analysis of post-contrast imaging showed that blood vessels were present and some appeared to be dilated. (See Appendix F for bSSFP images with pre- and post-Molday contrast agent).

3.6 References

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