Abstract 3543: Mesenchymal stromal cells and tumor-associated macrophages modulate adrenergic to mesenchymal state switching in neuroblastoma

Neuroblastoma (NB), a common solid tumor of children, can adopt a lineage-committed adrenergic (ADRN) or an immature mesenchymal (MES) tumor cell type, which differs in phenotype, epigenetic landscape, transcription factors, and core regulatory circuitries. These cell types can spontaneously interconvert, but the mechanism remains largely unknown. Here, we hypothesize that the tumor-associated macrophages (TAM) and cancer-associated fibroblast (CAF) within the tumor microenvironment contribute to pro-tumorigenic factors production and drive switching from ADRN to MES state in neuroblastoma. We first demonstrate the ADRN and MES lineage identity of NB cell lines by Western blot and compared the control and NB cell lines co-cultured with mesenchymal stromal cells (MSC), the precursor cell of CAF, and monocyte in transwell. Initial experiments demonstrated the morphology of NB cell line CHLA255 and CHLA136 showing increased spreading area and spindle shape in TAM-CAF co-cultures compared to control cells. As compared to control cells, monocytes and MSC-CAF co-culture induced the MES lineage markers SOX9 and Notch1 of NB cell lines that had low basal SOX9 expression, with a reduction in protein levels of ADRN lineage markers PHOX2B, and GATA3. Next, single-cell RNA-sequencing was utilized to analyze the expression of TAM and CAF, and MES- and ADRN-signature genes of NB cells in NB-TAM-CAF co-cultures. Single-cell analyses of TAM-CAF co-cultured CHLA255 and CHLA136 demonstrated enrichment of MES signature over time providing clear evidence of mesenchymal differentiation of NB cells within a TME-rich environment. Pathway enrichment analyses reveal enrichment of pathways associated with extracellular matrix (ECM) deposition and epithelial to mesenchymal of TAM-CAF co-cultured NB cell lines. Our results suggested that the presence of CAF and TAM in the tumor microenvironment drives neuroblastoma ADRN: MES switch akin to that observed in epithelial: mesenchymal switch in adult tumors. ADRN-MES state switching has been shown relevant for tumor relapse and therapy resistance. Our studies point to a therapeutic vulnerability by targeting cells and pathways activated in the NB-TAM-CAF axis. Citation Format: Meng-Hua Lee, Kevin Louault, Krinio Giannikou, Xiangming Ding, Alice Yanovsky, Jin-Seok Park, Yves A. DeClerck, Shahab Asgharzadeh. Mesenchymal stromal cells and tumor-associated macrophages modulate adrenergic to mesenchymal state switching in neuroblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3543.