Abstract 1921: Nonadherent human melanoma cell lines from lymph node metastases coexpress B cell markers and melanoma antigens
Authors: Hasskamp Joanne H. , Sharma Bhuvnesh K. , Zapas John L. , Elias Elias George 
Published: February 2011

Abstract
Introduction
Some atypical human melanoma cell lines are nonadherent to plastic culture flasks for unknown reasons. We have identified five nonadherent human melanoma cell lines derived from lymph node metastases that coexpress B cell markers CD20, CD19, CD21 and CD22 and melanoma antigens when grown in Iscove's-10% fetal bovine serum (FBS) growth medium.
Methods
The human melanoma cell lines were prepared from metastatic tumors of consenting patients by mechanical mincing into a single cell suspension. Human melanoma cell lines used in this study were grown in Iscove's medium supplemented with 10% FBS, L-glutamine and antibiotics in a humidified 37oC CO2 incubator and passaged at least 9 times, making it highly unlikely that any lymphocytes from the original tumor remained in the cultures. All human melanoma cell lines showed expression of at least one of the melanoma antigens Mart-1, gp100 (HMB45), TRP75 and melanoma-associated chondroitin sulfate proteoglycan (MCSP) as shown by flow cytometry on a FACS Calibur (BD Biosciences).
Two different antibodies, anti-human CD20 FITC clone 2H7 (BD Biosciences) and anti-human MS4A1/CD20 fluorescein clone 396444 (R& D Systems), substantiated the CD20 expression on the nonadherent melanoma cell lines. Isotype controls proved specific staining. Nonadherent melanoma cell lines were tested for CD19, CD20, CD21 and CD22, CD34 and CD133. Cells were stained for 15 minutes at room temperature protected from light, washed with 0.5% BSA-PBS, fixed in 1% paraformaldehyde-PBS and analyzed on the flow cytometer within 24 hours of fixation. Intracellullar staining for Mart-1, gp100 and TRP75 used Cytofix / Cytoperm and Perm / Wash reagents (BD Biosciences) according to the manufacturer's instructions, staining for 30 minutes at room temperature.
Results
The five nonadherent melanoma cell lines contained human melanoma cells that coexpressed CD20 and one of the four melanoma antigens Mart-1, gp100, TRP75 or MCSP. Percentages of double positive cells varied with CD20+ Mart-1+ tending to be the most frequent and expressed by the majority of the cells of the nonadherent melanoma cell lines. All five nonadherent melanoma cell lines expressed the B cell markers CD19, CD20, CD21 and CD22. However, none expressed the hematopoetic stem cell marker CD34.
The nonadherent human melanoma cell lines contained over 90% CD20+ cells and a small subpopulation of about 3% or less that coexpressed CD133. The immunophenotype of these atypical nonadherent melanomas suggests that CD20 may not be a stem cell marker but rather expressed with other B cell markers as a result of cell fusion events with B cells.
Conclusion
Nonadherent human melanoma cell lines derived from lymph node metastases that coexpress B cell markers CD19, CD20, CD21 and CD22 lend to speculation of their origin from cell fusion events.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1921.