Background High-risk neuroblastomas, including those with MYCN gene amplification, harbor abundant myeloid cells that suppress anti-tumor immunity and favor tumor growth. Macrophages lacking the inhibitory NF-κB p50 subunit adopt an M1-polarized, T-cell-activating phenotype. Multiple cancers grow slower in mice lacking p50, and colon cancer grows slower in mice lacking p50 specifically in myeloid cells.
Methods Tumor growth was monitored in mice inoculated subcutaneously with 9464D cells. To generate p50-IMC from p50-/-mice, marrow cells were lineage-depleted and then expanded in media with SCF, FL, and TPO, followed by transfer to M-CSF for one day. To generate p50-IMC from wild-type mice, marrow cells were nucleofected with a p50 sgRNA:Cas9 complex, followed by expansion in serum-free media. Mice received 5-fluorouracil on day 27, followed five days later by three doses of 1E7 p50-IMC via tail vein every three to four days. PD-1 antibody was administered twice weekly for four doses, starting on day 32. Azacytidine was administered 5 days per week alternating with ITF-2357 5 days per week, again starting on day 32. CD4 and CD8 antibody was given twice weekly starting on day 21. Tumor T cells were analyzed by flow cytometry.
Results We have now found that murine 9464D neuroblastoma cells, expressing high levels of exogenous human MYCN, also grow slower in syngeneic C57BL/6 (B6) p50(f/f);Lys-Cre mice, lacking p50 in macrophages and neutrophils, compared with p50(f/f) littermates. Slowed tumor growth in p50(f/f);Lys-Cre mice was associated with increased total and activated tumor CD4+ and CD8+ T cells, and depletion of both CD4+ and CD8+ T cells accelerated tumor growth. PD-1 checkpoint blockade or DNA methyltransferase and histone deacetylase inhibition further slowed tumor growth in p50(f/f);Lys-Cre mice. In addition, adoptive transfer of p50-IMC, generated either from the bone marrow of p50-/- B6 mice or via nucleofection of a p50 sgRNA:Cas9 complex into wild-type B6 hematopoietic progenitors, also slowed neuroblastoma tumor growth, following a dose of myelo-depleting 5-fluorouracil.
Conclusions These findings using a neuroblastoma model further validate the utility of targeting myeloid NF-κB p50 as an immunotherapy strategy for cancer therapy and demonstrate activity of p50-IMC generated by gene editing of syngeneic marrow cells, a cell product relevant to clinical translation. We have also developed means to efficiently gene edit p50 in human marrow CD34 cells, and have demonstrated the feasibility of generating p50-IMC from human induced pluripotent stem cells. We are currently evaluating the efficacy of these gene-edited human cells against human neuroblastoma in immune-deficient mice.
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