Expression of Multidrug Resistance Transporter ABCB5 in a Murine Model of Human Conjunctival Melanoma

Abstract

Conjunctival melanoma (CM) is a rare ocular malignancy with a high tendency to reoccur locally and with a high risk of metastatic disease. Metastases are often unresponsive to conventional treatment. Recently, an animal model was set up using human CM cells. Orthotopic xenografts from human CM were created by subconjunctival injection of three different CM cell lines into NOD.Cg-Prkdc^scid IL2rg^tm1Wjl/SzJ (NSG) mice. Subconjunctival injection of cultured CM cells led to excellent subconjunctival growth, but no metastases were found. When single-cell suspensions were obtained from the subconjunctival xenografts and passaged in vivo, all mice developed metastases. As recent findings indicate that cancer stem cells are linked to tumor recurrences, we used this new murine model to determine the expression of the stem cell marker ABCB5 during tumor progression. Expression of the ABCB5 protein was determined in three cell lines and during different stages of tumor development as observed in our model. All three cell lines contained a subpopulation of cells positive for ABCB5. During tumor development, expression of ABCB5 increased during phases of tumor expansion. Furthermore, expression of ABCB5 was increased in metastases. Using this model for CM, we were able to initiate metastatic spread and determine the expression of the stem cell marker ABCB5 during different stages of tumor development, identifying ABCB5 as a potential novel therapeutic target. This study illustrates the potential of our newly established murine model.

Process Development and Safety Evaluation of ABCB5+ Limbal Stem Cells as Advanced-therapy Medicinal Product to Treat Limbal Stem Cell Deficiency

“…In addition, in previously published studies, subconjunctival injection of 0.4 × 10 6 conjunctival melanoma cells (i.e. a dose similar to that of the ABCB5 + LSCs in the present study) to NSG mice resulted in ocular tumor development in 100% of the animals [75,76]. Together, these ndings con rm the ocular tumor susceptibility of the animal model used, which, in turn, counts against a tumor risk of treatment with ABCB5 + LSCs.…”

Evaluation of (fli:GFP) Casper Zebrafish Embryos as a Model for Human Conjunctival Melanoma

“…The results in vitro were expected as CRMM-1 and CM2005.1 harbor a B-RAF V600E mutation, while the CRMM-2 cell line contains an N-RAS Q61L mutation. 17,27 Vemurafenib was approved in 2011 by the Food and Drug Administration for treatment of unresectable melanoma harboring B-RAF V600E mutations 43 and is a potent agent for and P values were as follows: *P < 0.05, **P < 0.01, ***P < 0.001. For all groups: n ‡ 51. treatment of B-RAF V600E-positive melanoma.…”

“…Our group has shown that two of three available CM cell lines, CRMM1 25 and CM2005.1, 26 harbor a B-RAF V600E mutation, while the third, CRMM2, contains an N-RAS Q61L mutation. 17,27 We injected stable red fluorescently labeled (lentiviral tdTomato-blas) CM cells via different routes into the embryonic zebrafish. Thus, we determined the most effective engraftment strategy for the establishment of CM xenograft tumors in zebrafish and we observed distinct phenotypes after implantation of the three CM cell lines.…”

Targeting of the MAPK and AKT pathways in conjunctival melanoma shows potential synergy

“…These cells were grown in RPMI 1640 Dutch modified media (Gibco) supplemented with 10% FBS (Greiner Bio-one), 1% GlutaMAX and 1% Penicillin/Streptomycin (Gibco). Our previous studies [ 49 , 50 ] have shown that CRMM1 and CM2005.1 harbor a BRAF V600E mutation, and CRMM2 contains an NRAS Q61L mutation. These mutations were confirmed at Erasmus University Medical Center, Rotterdam in May 2016 by next generation sequencing (NGS).…”