Experimental studies, including the data presented above, clearly indicate that the biological functions of CS/DS chains depend on their fine structure. However, clinical data are still lacking to date to extend the validity of these in vitro experiments. For the first time, we have investigated the structure of CS/DS chains derived from small amounts of pathological samples using a sensitive mass spectrometric approach. To this end, CS/DS was purified and extensively degraded into disaccharides by a mixture of chondroitinases ABC, AC-I, and B enzymes. Interestingly, sulfated disaccharides were increased 5-fold in ESCC patient tumors compared to their normal counterparts (Fig. 5A). We further analyzed the type of sulfation on the disaccharides. Normal tissues contained approx. 82 % mono sulfated, 4-O-sulfated disaccharides and 18 % mono sulfated, 6-O-sulfated disaccharides, whereas cancer tissues contained approx. 65 % mono sulfated, 4-O-sulfated disaccharides and 35 % mono sulfated, 6-O-sulfated disaccharides (Fig. 5B). Disulfated disaccharides within CS/DS chains are less abundant components that have been associated with biological functions. We present data showing that these disaccharides represented approx. 7 % of the total sulfated disaccharides in normal esophageal tissues. Their relative content in all cancer biopsies decreased approx. 4-fold compared to normal tissues (Fig. 5C).
