Neuroblastoma (NB) is the most common solid cancer in children outside of the skull and it still kills about 40% of patients. There is increasing evidence that the tumor microenvironment promotes resistance of NB to chemotherapy. In particular, Tumor-Associated Macrophages (TAMs) promote NB growth and resistance. However, there are two fundamental gaps in our knowledge of this interaction: 1) We do not know which molecular mechanisms mediate TAM pro-tumoral effects and consequently we are unable to exploit such mechanisms for new therapeutic purposes; 2) We have not identified a “systemic” parameter that reflects the degree of TAM infiltration in the primary tumor, and consequently we cannot identify which subsets of patients would particularly benefit from an anti-TAM therapy. Our preliminary data support a role for microRNAs (miRs) within exosomes as responsible for the increased NB proliferation and drug resistance through the direct targeting of TP53, the most frequently dys-regulated gene in human cancers and with a well established role in multi-drug resistance in NB. Specifically, NB cells secrete exosomal miR-21, which is up-taken by surrounding macrophages and can bind to Toll-like receptor 8 (TLR8), triggering TLR8 activation in macrophages. As a consequence of this activation, we showed up- regulation of miR-155, -487a and -597, all predicted to target TP53. We also engineered a nanoparticle coated with anti-CD163 antibody to specifically silence miR-155 in TAMs (that are CD163+). Finally, we were able to develop a modified protocol that successfully isolates purer exosomes (meaning with lower protein contaminants) both from cell supernatants and from patients’ plasma. With this protocol we isolated CD163+ exosomes (released by TAMs) from the plasma of NB patients and healthy donors, and showed increased levels of exosomal miR-155 in the plasma of NB patients compared to healthy donors. Therefore, we hypothesize that NB cells, by secreting exosomal miR-21 that binds to TLR8 in surrounding TAMs, induce the secretion of exosomal miR-155, -487a and -597 by TAMs and these miRs are transferred back to NB cells, where they silence TP53 and increase NB multi-drug resistance. We also hypothesize that targeting these miRs will restore sensitivity to chemotherapy. Finally, we believe that the levels of TAM-derived exosomal miRs will reflect the degree of TAM infiltration in the primary tumor and will correlate with clinical outcome measures. We will investigate these hypotheses in 3 specific aims: 1) a study of the mechanisms by which exosomal miRs induce resistance to therapy in NB; 2) an assessment of the therapeutic potential of targeting NB and TAM-derived exosomal miRs to overcome NB resistance; 3) a determination of TAM-derived exosomal miRs as indicators of TAM-infiltration in the primary tumor and of clinical outcome measures. The successful completion of this research will identify new molecular targets for NB, identify subsets of patie...