These tumors are morphologically composed of heterogeneous cells, ranging from smaller non-vacuolated spindly-shaped cells to large cells with prominent vacuoles; the latter are referred to as the physaliferous cells. Furthermore, ElHeliebi et al. found four candidate genes that were possibly responsible for the heterogeneity in cell development by phenotype-specific GANT61 analyses of the small non-vacuolated and the large physaliferous cells within two independent chordoma cell lines. Therefore, we postulate that a detailed ultrastructural testing of the vacuoles, observed in this chordoma cell type, represents an important step towards an improved understanding of their tumor biology. Currently, the most efficient way to obtain information on cell interactions when using electron microscopy is by combining high pressure freezing with electron tomography. The combination of both techniques enables us to visualize the dynamic process of the cell compartments at any known time point, in three dimensions, as close as possible to the native state; furthermore, it is possible to obtain interactions with reduced artifacts which are often generated by chemical fixation. Detailed ultrastructural analysis offers valuable information on the biological behavior of chordomas. In addition to the ultrastructural characterization of the vacuoles from chordoma, as early as 1968 Erlandson et al. had already showed, an accumulation of complexes where mitochondrial membrane was tightly associated with the endoplasmic reticulum, a formation termed MAM. This conspicuous surrounding area of most mitochondria composed by the rough ER seems to be very specific to chordoma. The main function of the MAM complex is to enable the transfer of lipids and calcium between the two organelles, and it is also involved in mitochondrial physiology and apoptosis. Based on the number and size of MAM complexes and due to the high number of vacuoles, we investigated the mechanisms of intracellular calcium signaling via stimulation through acetylcholine to promote Ca2+ release, and the sphingolipid metabolism in form of lipid composition. Ca2+ signaling plays a role in many cellular processes, however, altered expression of specific Ca2+ _channels and pumps is a characterizing feature of some cancers. Changes ascribed to sphingolipid metabolism characterize different cancers and are important for maintenance of the cancer phenotype. Ceramides have been known to regulate programmed cell death.. Glycosylceramide synthase catalyzes the glycosylation of ceramide to glycosylceramids, which have been found to involve many cellular processes such as cell proliferation and tumor metastasis. Drug resistance has also been strongly associated with GlyCer. Our aim was to find a new perspective on the biology of chordoma that would open up the field for completely new therapeutic approaches with regard to lipids and Ca2+ pathways to finally overcome thus far untreatable chordoma tumors. Today, the quality of microscopic technology increased to very high level of specificity.