## 6 Farber disease
### MALDI Non-Imaging MS Analysis of GSLs in Various Cell Cultures
#### 3.6.1 GSLs in Breast Cancer Cell Lines  
Breast cancer is a type of cancer that originates in the breast tissue, typically in the ducts or lobules. It represents the most frequent form of cancer among women internationally, and although it can also affect men, it is a rare finding among men [245]. Currently, intense research is taking place to find means of prevention, diagnosis, and treatment for breast cancer, and the discovery of the molecular pathways involved in its development is a crucial step in this direction. GSLs, with the previously mentioned properties, have been shown to contribute to immune evasion (GSLs are thought to suppress immune responses and promote tumor immune escape) [246; 247; 248; 249] and to tumor progression (tumor growth and tumor metastasis) and may interfere with drug resistance (chemoresistance) of certain tumor types. The particular expression of certain GSLs has been shown to be associated with breast cancer diagnosis and with its prognosis [248; 249; 250; 251; 252; 253; 254; 255; 256; 257; 258; 259; 260; 261; 262; 263; 264]. The use of MALDI MS is crucial for identifying a specific GSL profile for each type and subtype of cancer, and a tremendous amount of further work is needed for the correct and detailed identification of GSLs, in the hope of finding new options for diagnosing and treating this disease. Cavdari et al. [265] performed an analysis of GSLs expressed by neuroectoderm-derived cell lines, to determine the pattern of \(O\)-acetylated native ganglioside species, considering that previous studies [260; 266; 267; 268] have shown that in neuroectodermal-derived cancer cells, ganglioside GD2 and GD3 overexpression participates in tumor aggressiveness. Whereas there exists limited knowledge regarding \(O\)-acetylation mechanisms, in cancer research, gangliosides (specifically GD3 and GD2) are believed to be tumor-associated carbohydrate antigens (TACA), and the \(O\)-acetylated form of GD2 (O-Ac-GD2) has been found to be exclusively related to cancer cells and tissues. For this research, the authors used commercially available cell lines for breast cancer: Hs587T, SUM159 PT, MDA-MB-231, and MCF7, and breast cancer cell lines overexpressing GD3 synthase. The cell lines were cultured, and for an MS analysis they were extracted and purified with methods designed to preserve the \(O\)-acetylation of the GSLs. The separated acidic GSL fraction was analyzed on an MALDI-QIT_TOF (Shimadzu AXIMA Resonance) MS, using THAP as a matrix solution, containing 0.1 M hydrated di-ammonium hydrogen citrate mixture (\(2/1;v/v\)), in negative ion mode. The mass spectra for the Hs587T cell line showed the presence of GM3 (at \(m/z\) 1263.8, d18:1, C24:0), GM2 (at \(m/z\) 1354.8 d18:1, C16:0; \(m/z\) 1438.9, d18:1, C22:0, and \(m/z\) 1466.9, d18:1, C24:0), GM1 (at \(m/z\) 1516.8, d18:1, C16:0; \(m/z\) 1629.0, d18:1, C24:0), OAc-GM1 (at \(m/z\)1671, d18:1, C24:0), OAc-GD2 (at \(m/z\) 1670, d18:1, C16:0 \(\left[\rm{M-2H_{2}O-H}\right]^{-}\)), and traces of GD2, OAc-GD3, and OAc-GT3. For the MDA-MB-231 cell line (GD3S+ clone #4), the results showed a similar pattern of gangliosides, but for the \(O\)-acetylated species, a cell-dependent pattern was observed, with OAc-GD3 (at \(m/z\) 1438.8, with Cer (d18:1/14:0)), OAc-GD2 (at \(m/z\) 1782 with Cer (d18:1/24:0)) and OAc-GT3 (at \(m/z\) 1852.0, with Cer (d18:1/24:0)), identified as the main \(O\)-acetylated ganglioside species as dehydrated negative ions. After performing MALDI MS on all the cultured cell lines, it was found that all the cell lines express GM1, GM2, GM3, and GD2. GD3, GT3, and GT2 were expressed only in the MDA-MB-231 GD3S+ clones, with GD3 synthase overexpression being associated with a dramatic change in ganglioside composition towards b- and c-series gangliosides [204]. For the \(O\)-acetylated ganglioside species, OAc-GD2 was found in the Hs587T and SUM159 PT cell lines, OAc-GD3 in Hs 587T, SUM159 PT and MDA-MB-231 GD3S+ clone cell lines and OAc-GT3 and OAc-GT2 were expressed in the MDA-MB-231 GD3 synthase overexpressing clones.