Background: Tumor-associated macrophages (TAMs) with alterations in patterns of pro-inflammatory genes are associated with poor prognosis in neuroblastoma (NB). TAM can promote tumor growth and create and anti-inflammatory environment, while expression of the immune checkpoint protein PDL1 can act to block T-cell response via the PD1-PDL1 axis. We set out to identify compounds that inhibit tumor-promoting effects of TAM and assess their efficacy in enhancing immune checkpoint therapy using a transgenic MYCN non-amplified neuroblastoma murine model (NB-Tag).
Methods: Human and murine neuroblastoma cell lines (NBT2 and NBT3L) were used for in vitro studies. For in vivo tumor growth models, combinations of cyclophosphamide, topotecan, trametinib, anti-CTLA4, and anti-PD1 therapies were used in NB-Tag transgenic, transplantable subcutaneous (NB-SQ), and orthotopic models.
Results: Co-culture of M2-like macrophages with human or murine neuroblastoma cell lines increased tumor proliferation and upregulated MYC expression in vitro. Trametinib, a MAPK inhibitor, was identified among a small drug screen with the exceptional ability to inhibit the tumor-proliferative advantage provided by macrophages in the co-culture system. Treatment of 15 week-old NB-Tag mice (visible tumor by MRI) with trametinib after chemotherapy administration significantly impaired tumor regrowth (volume four weeks post-chemo, 491 vs. 42 mm3, p=0.037). Trametinib treatment also showed similar results in NB-SQ model. Further immune checkpoint blockade of NB-SQ mice with anti-CTLA4 and anti-PD1 in combination with Trametinib treatment post chemotherapy significantly increased survival compared to controls (OS 60% vs. 0%; p<0.0001), and led to long term cures. Tumors and macrophages collected from mice treated with Trametinib showed effective block in phosphorylation of ERK, and attenuation of COX2 expression in macrophages. Transgenic animals that grew despite combination treatment showed STAT3 phosphorylation suggesting this pathway as an escape mechanism.
Conclusions: Our results provide strong evidence that blocking macrophages ability to promote tumor growth through targeted therapies combined with checkpoint blockade can significantly inhibit tumor formation. These findings indicate opportunities to enhance antitumor immunity with the potential to produce durable clinical responses in children with neuroblastomas.