Targeted liposomal doxorubicin/ceramides combinations: The importance to assess the nature of drug interaction beyond bulk tumor cells

One of the major assets of anticancer nanomedicine is the ability to co-deliver drug combinations, as it enables targeting of different cellular populations and/or signaling pathways implicated in tumorigenesis and thus tackling tumor heterogeneity. Moreover, drug resistance can be circumvented, for example, upon co-encapsulation and delivery of doxorubicin and sphingolipids, as ceramides. Herein, the impact of short (C6) and long (C18) alkyl chain length ceramides on the nature of drug interaction, within the scope of combination with doxorubicin, was performed in bulk triple-negative breast cancer (TNBC) cells, as well as on the density of putative cancer stem cells and phenotype, including live single-cell tracking. C6- or C18-ceramide enabled a synergistic drug interaction in all conditions and (bulk) cell lines tested. However, differentiation among these two ceramides was reflected on the migratory potential of cancer cells, particularly significant against the highly motile MDA-MB-231 cells. This effect was supported by the downregulation of the PI3K/Akt pathway enabled by C6-ceramide and in contrast with C18-ceramide. The decrease of the migratory potential enabled by the targeted liposomal combinations is of high relevance in the context of TNBC, due to the underlying metastatic potential. Surprisingly, the nature of the drug interaction assessed at the level of bulk cancer cells revealed to be insufficient to predict whether a drug combination enables a decrease in the percentage of the master regulators of tumor relapse as ALDH+/high putative TNBC cancer stem cells, suggesting, for the first time, that it should be extended further down to this level.