Abstract 3622: Differential effect of liposomal C6-ceramide/doxorubicin targeted to nucleolin and conventional combinations against triple negative breast cancer stem cells
Authors: Cruz Ana F. , Fonseca Nuno A. , Gonçalves Nélio , Moura Vera , Simões Sérgio , Moreira João Nuno 
Published: August 2019

Abstract
The interconversion between cancer stem cells (CSCs) and non-stem cancer cells (non-SCCs) in response to environmental stimulus, has urged the need to identify markers common to both cell sub-populations. This is the case of cell surface nucleolin, a target for drug delivery, offering the opportunity to target both cell populations as previously demonstrated (Fonseca, Biomaterials 2015). Herein, it is hypothesized that the intracellular delivery of the developed liposomal synergistic combination of doxorubicin (DXR) and the sphingolipid C6-ceramide (C6), functionalized with a nucleolin-binding peptide, [F3]L-DC6, will imbalance the CSC niche of triple-negative breast cancer (TNBC), relative to a clinically used combination of DXR and cisplatin (Cis). Cytotoxicity against (bulk) TNBC cell lines (MDA-MB-231, MDA-MB-468 and HS-578T) was assessed by the rezasurin assay. The impact over the putative CSCs population (ALDHhigh, as determined by the Aldefluor assay) was evaluated by flow cytometry. Moreover, assessment of the effect of each drug combination on the in vitro stemness properties of TNBC cells (the mRNA levels of pluripotency transcripts OCT4 and NANOG and mammosphere-forming capacity) was also performed. The intracellular delivery of the DXR:C6 (1:1 molar ratio) combination provided by the formulation targeted to nucleolin ([F3]L-DC6) enabled a significant increase in bulk cell death, compared with the counterpart single drug formulation. In fact, only the targeted liposomal DXR:C6 combination enabled a 90% death in the case of MDA-MB-231 cells, for an exposure as short as 4 h. This was in contrast with the lack cytotoxic benefit of the (non-liposomal) DXR:Cis combination over each drug alone, against each one of the TNBC cell lines used. Despite increasing concentrations of the DXR:Cis combination, enabled higher (bulk) cell death, the percentage of ALDHhigh viable cells increased by 1.5-fold relative to the untreated control, in the case of MDA-MB-468 cells. Interestingly, the opposite effect was observed for the DXR:C6 combination targeted to nucleolin, where increasing concentrations enabled higher (bulk) cell death, along with a 5-fold decrease of the percentage of viable ALDHhigh putative CSCs (relative to untreated control). These observations were concomitant with alterations of stemness properties favoring the latter. Overall, the enhanced efficacy of the synergistic DXR:C6 combination targeted to nucleolin, enables a significant decrease of the percentage of chemoresistant triple negative breast CSC, besides non-SCCs, in contrast to observations with clinically used drug combination. Thus, this novel strategy offers the potential to address tumor recurrence and drug resistance associated with CSC, in an unmet medical need as TNBC.
Citation Format: Ana F. Cruz, Nuno A. Fonseca, Nélio Gonçalves, Vera Moura, Sérgio Simões, João Nuno Moreira. Differential effect of liposomal C6-ceramide/doxorubicin targeted to nucleolin and conventional combinations against triple negative breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3622.