## 6 Farber disease
### MALDI Non-Imaging MS Analysis of GSLs in Various Cell Cultures
#### 3.6.1 GSLs in Breast Cancer Cell Lines
It was found that the highest amount of \(O\)-acetylated gangliosides corresponded to overexpressing GD3 synthase clones, in a cell-dependent manner (by integrating the intensity of individual signals detected on MALDI-QIT-TOF): 50% in MDA-MB-231 GD3S+ clone#11, 22% in MCF-7 GD3S+, and 18% in MDA-MB-231 GD3S+ clone#4. Furthermore, it was found that overall, OAc-GD3 was the most expressed ganglioside species, followed by OAc-GD2 and OAc-GT3. Cavdarli et al. [267] emphasized the importance of ganglioside diversity and the presence of \(O\)-acetylated gangliosides in breast cancer, and also the importance of considering \(O\)-acetylated gangliosides as a possible target for developing new specific therapies for the disease. While the findings show promise, additional studies are required to unravel the intricate pattern of ganglioside expression in breast cancer. Using a TOF/TOF 5800 MALDI System (Sciex, Vaughan, Canada), and DHB as a matrix for MALDI MS, in positive ion mode, Liang et al. [257] determined if several breast cancer cell line subtypes (MDA-MB-231, MDA-MB-468, and MCF7) with or without GD3-synthase expression exhibited distinct profiles for GD2 or GD3 expression. The GSLs were extracted by successive sonication steps in solvents (chloroform/methanol, isopropanol/hexane/water) and Folch's partitioning, with the upper phases collected, combined, evaporated, and solubilized in water and applied to a Sep-Pak C18 cartridge, then analyzed via HPTLC. For MALDI profiling, the extracted GSLs were permethylated and dissolved in acetonitrile solution, then applied to the 2,5 DHB matrix. In the MDA-MB-231 cell line, the major GSLs were GM1, GM2, and GM3 (a-series gangliosides), while b-series gangliosides (GD1, GD3) and _globo_-series GSLs (Gb4, MSGb4 (monosialoGb5), DSGb5 (disialoGb5)) were detected at low levels. In parallel, GSLs from RFP (red fluorescent protein) mock-transfected cells and GD3-synthase-overexpressing cells were also analyzed, with MALDI MS confirming that GD3S overexpression resulted in the upregulation of GD2 (9.8% in the wild-type MDA-MB-231 cell line, 14.7% in RFP cells, and 60.2% in the GD3S+ cell line) and GD3 (7.7% in wild-type MDA-MB-231 cell line, 6.8% in RFP cells, and 25.8% in GD3S+ cell lines) and downregulation of GM3, GM2, GM1, GD1, and Gb4/(n)Lc4. For the MDA-MB-468 cell line, following the same protocol, it was found that the main GSLs expressed were GM1 and GD3, with low levels of GM3, GT3, GD1, and Fucosyl-Lc4 detected. While targeting the GD3S+ cell lines again, there was no significant difference in GD2 determination, and only GD3 was found to be upregulated (from 77.4% in the wild-type MDA-MB-468 cell line, and 70.2% in the RFP control line, to 87.2% in the GD3S+ cell line), and also an increase in GT3 and GD1 ganglioside levels. In MCF7 cells, GM2 was the only highly expressed GSL in the wild-type population of cells, and Gb4, Gb5, globo-H (neutral GSLs) and GM3, MSGb5, and DSGb5 (sialylated GSLs) were also detected. For the GD3S+ variant of these cells, an upregulation of GD2 was observed (from 0.9% in wild-type cells MCF7 and 1.1% in RFP control cells, to 57.3% in GD3S+ cells), and only a minor increase in GD3 (from 0.7% in wild-type MCF7 cells and 0.8% in RFP control cells, to 3.7% in GD3S+ cells). Furthermore, an increase in GD1 was noted, along with various globo- and lacto-series GSLs: Gb4/(n)Lc4, Gb5, globo-H, MSGb5, DSGb5. The results obtained by Liang et al. [257] confirm that GSL expression in cell lines expressed in breast cancer is cell-type dependent, with a complex and intricate pattern for each cell line studied. Examining GD3-synthase-overexpressing cells, it was noted that GD2/GD3 GSLs constitutes a vital element that maintains the stem cell phenotype of breast cancer cells and amplifies the migration, adhesion, and clonogenic potential of malignant cells, thus being an important target for a possible therapy. Using a 4800 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA) in positive reflectron mode, and with 2.5 DHB as a matrix, Dewald et al.