Abstract A29: Differential expression of the gangliosides GD3 and GD2 in canine and human osteosarcoma cell lines: An immunotherapy target

Introduction: The disialyl gangliosides GD2/GD3 have been implicated in the enhancement of malignancy in a number human and animal caners (Milner et al Vet Immunology 2006). Moreover, pediatric research trials targeting GD2 osteosarcoma (OSA) surface antigens using humanized mouse antibodies is currently underway. While the coexpression of GD3 and GD2 have been documented in human OSA it has been not reported in canine OSA. Canine OSA is an accepted phenotypic and genotypic large animal model for the study of human OSA. We report on the co-expression of GD3/GD2 in three canine and one human OSA cell lines. Material and Methods: Four canine OSA cell lines (POS, HMPOS, COS31, and Doug) and one human OSA cell line (MG63) were trypsinized from T75 flasks and counted using trypan blue exclusion dye. Live cells (1x10 6 ) were spun down at 250 x gravity. Supernatant was removed and cell pellet mixed with 1ml PBS wash solution. Samples were then centrifuged, supernatant removed, and cells were washed two more times. Primary antibodies GD2 antibody (clone 14G2a, Santa Cruz) and GD3 Mel-1 (clone R24, Covance) and isotype controls were added to samples which were then incubated on ice in the dark for 30 minutes. Cells were again washed in PBS wash solution. Samples were blocked using normal goat serum, washed and then incubated with detection antibody goat anti-mouse IgG F(ab9)2 conjugated to Alexa Flour 488® or Alexa Fluor 647 Goat anti-mouse IgG2a [Molecular Probes, Eugene, OR USA] ) for 30 minutes on ice in the dark. Cells were then washed in wash buffer and re-suspended in 1ml of PBS wash solution. Samples were read at 10,000 events/tube on FACScan (BD, Franklin Lakes, NJ USA). Immunofluorescence was documented using confocal microscopy was used to establish the co-expression of GD3 and GD2 on the cell surface. Results: All cell lines, expressed varying levels of GD3 and GD2 on the cell surface. The canine OSA cell line appeared to express more GD3 (12.54%) on the surface compared to the human MG63 cell line (0.02%). Remarkably, POS, the primary tumor cell line of HMPOS, expressed very low levels of GD3 (4.65%) and GD2 (0.24%), whereas the metastatic line (HMPOS) expressed higher GD3 (25.4%). All cell lines had cells that expressed either GD2 or GD3 but not both. The human MG63 cell line expressed the highest levels of GD2 alone (68.4%) compared to canine cells (average 0.58%). The average co-expression of GD2/GD3 was 48.1% for all cell lines. Discussion: This is the first report of the expression of both GD3 and GD2 in canine OSA cells. The related cell lines POS and HMPOS showed significant variation in expression, with HMPOS (the metastatic line) showing increased expression of GD3. The differential expression of GD3/GD2 on these two cell lines may indicate a phenotype change associate with metastatic potential. The highest co-expression of GD2/GD3 was found in MG63 and COS31, but was also found in HMPOS and Doug. Current investigation includes expression of GD2 & GD3 in osteosarcoma stem cell populations and invasive potential. Conclusion: Results from this study will provide data to investigate the expression profiles of GD2/GD3 and its role in metastasis as well as targeting OSA tumor antigens. Citation Format: Rowan J. Milner, Noaki Chimura, Kristina D. Bowles, Marc Salute. Differential expression of the gangliosides GD3 and GD2 in canine and human osteosarcoma cell lines: An immunotherapy target. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A29.