ANAKIN: Advanced Nanotechnology to Assist Keeping the tumor microenvironment Involved in cancer Neutralization

Anticancer nanoparticles (NPs) often fail to translate clinically. Aside from a rapid clearance by the immune system, one of the major mechanisms is the unspecific uptake of NPs by stromal cells – mainly Cancer Associated Fibroblasts (CAFs) – especially in desmoplastic cancers. ANAKIN project aims at overcoming the immune and the stromal barriers to NPs delivery in desmoplastic cancers by the development of immune system-invisible cancer cells-derived biomimetic NPs to vehicle to CAFs an mRNA encoding for the tumor-selective cytotoxic agent TRAIL (Tumor necrosis factor-Related Apoptosis-Inducing Ligand), that should trigger cancer cells apoptosis through an exosomes-mediated mechanism. Inserted in ANAKIN project, this work has two objectives: 1) the development of a CAFs model and 2) the optimization of CAFs transfection to maximise TRAIL production. To resemble CAFs population we treated NIH3T3 fibroblasts with TGF-β (Transforming Growth Factor beta), the most potent inducer of fibroblasts’ cancer-associated phenotype, and we confirmed the cellular differentiation by analysing two main CAFs features: the overexpression of the CAFs marker α-SMA (alpha Smooth Muscle Actin), analysed by Western Blot, and CAFs metabolic reprogramming towards aerobic glycolysis, analysed by Seahorse. To optimise TRAIL production in CAFs we compared pDNA transfection and mRNA transfection. As both in case of pDNA and mRNA TRAIL is fused to the fluorescent tag EGFP (Enhanced Green Fluorescent Protein), we evaluated the transfection efficiency by flow cytometry. By flow cytometry we also assayed transfection-associated toxicity. To validate the suitability of the fusion protein in terms of retention of TRAL apoptotic activity and selective induction of apoptosis in cancer cells, we also transfected 4T1 cancer cells – that we selected as a model for desmoplastic tumors – and CAFs with EGFP and EGFP-TRAIL mRNAs and we evaluated the cellular apoptosis by the annexin V/DAPI assay. According to the differentiation studies, TGF-β activates NIH3T3 fibroblasts into CAFs by inducing the overexpression of α-SMA and a more energetic phenotype due to an enhanced glycolysis. CAFs transfection studies demonstrated that mRNA transfection solves the problems of low transfection efficiency and high toxicity associated to pDNA transfection. For this reason, we selected the mRNA for the following studies. The transfection of 4T1 cancer cells and CAFs with EGFP and EGFP-TRAIL mRNAs revealed that TRAIL fusion to the fluorescent tag probably doesn’t affect TRAIL cytotoxic activity and that 4T1 cells, but not CAFs, are TRAIL-sensitive. The next steps will be the isolation and characterization of transfected CAFs-released exosomes to demonstrate the vesicular localization of TRAIL, and the validation of the apoptotic activity of TRAIL+ exosomes in vitro.