Abstract 3914: In vivo label-free examination of early-stage melanoma formation.
Authors: Degan Simone , Wilson Jesse W. , Gainey Christina S. , Kent Julie , Reddick Traci L. , Nelson Kelly , Selim Maria A. , Warren Warren S. 
Published: August 2014

Abstract
Rationale. One of the most lethal forms of skin cancer is the cutaneous melanoma that develops from melanocytes. Distinguishing between benign and malignant pigmented lesions is difficult and often requires excision for pathological examination. In most malignant melanoma, an early genetic alteration of the melanocytes is a mutation of the BRAF oncogene. We have used a pre-clinical mouse model that allows conditional induction of the BRAF oncogene and deletion of Pten tumor suppressor in melanocytes. Upon induction, these animals develop melanomas that reproduce many of the histological and molecular changes observed in humans. With pump-probe imaging in vivo we have non-invasively detected the early modifications that harbor melanoma formation. We have investigated the sub-cellular expression of melanin as an indicator of tumor progression along with the morphological modifications that characterize the pigmented malignant lesions. Methods. Transgenic mice (Tyr::CreER(Tg/+); BRaf(CA), Pten(lox/lox)) were treated topically 4-hydroxytamoxifen (4-OHT: 1.5uL of 5mM) to induce conversion of BRaf(CA) to BrafV600E and Pten silencing (n=4). In vivo pump-probe microscopy was performed to acquire 3D, noninvasive images of the pigmented lesions, using the melanin pigment itself for imaging contrast. Images were acquired 2-day after 4-OHT application, at 2 and 4 weeks. C54 mice (n=2) were used as control. Pump-probe was combined with multiphoton autofluorescence. In vivo confocal microscopy was performed for morphological evaluation and blood flow detection. Afterwards, mice were euthanized, the skin was resected and sections were evaluated after staining with hematoxylin-eosin (HE). Results. Pigmented lesions were detectable by pump-probe microscopy two days after the treatment. The second scan (2-week) showed strong pigmentation present both, superficially and around the canal root of the hairs. The subsequent scan (4-week) showed the invasive nature of the lesions with altered cellular architecture resembling vertical growth melanomas. Pigmentation and atypical cellular proliferation was also present in the deeper layers of the dermis indicating the invasiveness of the tumor. These findings were confirmed by histological examination of the HE slides. In vivo confocal microscopy showed prominent convoluted vessels with large caliber and high blood flow confirming the malignant nature of the lesions. The control subjects did not show the cellular formations typical of the melanocytic lesions. Conclusions. Pump-probe laser microscopy detected in vivo and label-free the morphological changes that take place in melanoma formation. In contrast to the control subjects, melanocytic lesions were easily identifiable 2- day after the treatment with 4-OHT. This new non-invasive diagnostic approach is helpful in identifying early stage cutaneous melanomas.
Citation Format: Simone Degan, Jesse W. Wilson, Christina S. Gainey, Julie Kent, Traci L. Reddick, Kelly Nelson, Maria A. Selim, Warren S. Warren. In vivo label-free examination of early-stage melanoma formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3914. doi:10.1158/1538-7445.AM2013-3914