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 patients who can benefit from an anti-TAM therapy, and will increase the number of saved lives of children affected by NB.

Public Health Relevance

Neuroblastoma (NB) is the most common type of solid cancer in children outside of the skull. This study will investigate whether Tumor-Associated Macrophages (TAMs) surrounding NB cells induce resistance to chemotherapy by transferring specific microRNAs (miRs) within extracellular vesicles called exosomes, and how this mechanism can be exploited for therapeutic purposes. Taking advantage of the unique set of samples collected by the New Approaches to Neuroblastoma Therapy (NANT) consortium, this project will also assess whether circulating exosomal miR levels correlate with the degree of TAM infiltration in the primary tumor and with measures of disease burden and clinical outcome in recurrent/refractory neuroblastoma.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA219024-01A1
Application #
9470761
Study Section
Clinical Oncology Study Section (CONC)
Program Officer
Kondapaka, Sudhir B
Project Start
2018-01-01
Project End
2022-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Children's Hospital of Los Angeles
Department
Type
DUNS #
052277936
City
Los Angeles
State
CA
Country
United States
Zip Code
90027