Журнал аналитических и биоаналитических методов

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Ceramide and Sphingosine-1-Phosphate/Sphingosine act as Photodynamic Therapy-Elicited Damage-Associated Molecular Patterns: Release from Cells and Impact on Tumor-Associated Macrophages

Mladen Korbelik, Judit Banáth, Wei Zhang, Fred Wong, Jacek Bielawski and Duska Separovic

A recent finding showed that ceramide and sphingosine-1-phosphate (S1P) become exposed on the surface of cells treated by photodynamic therapy (PDT) and acquire the capacity to act as danger-associated molecular patterns (DAMPs). To explore this further, the present study examined whether ceramide and S1P can be released from PDTtreated cells and investigated changes in the levels of these sphingolipids in tumor-associated macrophages (TAMs) left in contact with PDT-treated tumor cells. Massspectroscopy-based analysis detected increased levels of C16- ceramide and dihydroC16-ceramide in media supernatants from SCCVII cells collected three hours after they were treated by PDT, compared to untreated cell supernatants. While no release of S1P was detected, elevated levels of its precursor sphingosine were found in the supernatants of PDT-treated cells. The co-incubation of TAMs-containing primary cultures derived from mouse SCCVII tumors with PDT-treated SCCVII cells was followed by ceramide and S1P analysis in these cells based on staining with specific antibodies and flow cytometry. Levels of both ceramide and S1P as well as inflammasome protein NLRP3 were found to rise in TAMs when they were co-cultured with PDT-treated SCCVII cells, while no significant change was seen with cancer cells. Such changes were induced also in TAMs incubated with supernatants from PDT-treated cells. The findings of the present study affirm the potential of sphingolipids including ceramide, S1P, and sphingosine to act, either exposed on cell surface or released in the microenvironment, as DAMPs in the response of tumors to PDT.