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Neuroblastoma (NBL) accounts for a disproportionate number of deaths among childhood malignancies despite intensive multimodal therapy that includes antibody targeting disialoganglioside GD2, a NBL antigen. Unfortunately, resistance to anti-GD2 immunotherapy is frequent and we aimed to investigate mechanisms of resistance in NBL. GD2 expression was quantified by flow cytometry and anti-GD2 antibody internalization was measured using real-time microscopy in 20 human NBL cell lines. Neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were performed on a subset of the cell lines (n = 12), and results were correlated with GD2 expression and antibody internalization. GD2 was expressed on 19 of 20 NBL cell lines at variable levels, and neutrophil-mediated ADCC was observed only in GD2-expressing cell lines. We found no correlation between level of GD2 expression and sensitivity to neutrophil-mediated ADCC, suggesting that GD2 expression of many cell lines was above a threshold required for maximal ADCC, such that expression level could not be used to predict subsequent cytotoxicity. Instead, anti-GD2 antibody internalization, a process that occurred universally but differentially across GD2-expressing NBL cell lines, was inversely correlated with ADCC. Treatment with endocytosis inhibitors EIPA, chlorpromazine, MBCD, and cytochalasin-D showed potential to inhibit antibody internalization; however, only MBCD resulted in significantly increased sensitivity to neutrophil-mediated ADCC in 4 of 4 cell lines in vitro. Our data suggest that antibody internalization may represent a novel mechanism of immunotherapy escape by NBL and provide proof-of-principle that targeting pathways involved in antibody internalization may improve the efficacy of anti-GD2 immunotherapies.

The individual contribution of tumor-associated macrophages (TAM) and cancer-associated fibroblasts (CAF), and their precursors mesenchymal stromal cells (MSC) to a pro-tumorigenic and immune-suppressive tumor microenvironment (TME) in neuroblastoma (NB) is well known. However, whether and how they could cooperate has not been studied in this cancer. This important question, in the context of current immunotherapies for NB, has been addressed here in complementary studies evaluating the TME landscape in primary human NB tumors and examining the production of cytokines/chemokines and the activation of signaling pathways in NB cells in the presence of human monocytes and MSC-CAF in vitro and in vivo. A multiplex immunohistochemistry evaluation of 17 untreated human NB tumors demonstrated the presence of MSC-CAF and TAM representing between 0.5% to 7.4% and 0.4% to 8.4% of the total cell population respectively. MSC-CAF were predominantly present in the stroma around NB cells, while TAM diffusely infiltrated tumors. There was a paucity of infiltrating T cells and NK cells (0.7% and 0.1% of total cells, respectively) in all tumors. MSC-CAF promoted in vitro the survival of human monocytes and TAM through their production of IL6 and soluble IL6 receptor. When co-cultured with NB cells, MSC-CAF and monocytes increased the production of pro-tumorigenic cytokines/chemokines like TGFβ1, IL6, IL8, IL10, CCL2/MCP-1 and induced a high expression in MYCN amplified NB cells and TME cells. In contrast they did not stimulate the production of anti-tumorigenic cytokines like IFNγ, TNFα and IL12. Among these multiple cytokines, we demonstrated the central role of TGFβ1 which induced the expression of IL6 in NB cells and in MSC-CAF. Mechanistically, we demonstrated that production of IL6 in NB cells was dependent on the activation of NFκB by TGFβ1 via TAK1 and SMAD2 signaling pathways. In contrast, in MSC, the production of IL6 was dependent on the transcriptional activity of SMAD2/SMAD4 but independent of NFκB. Moreover, the significant increase in TGFβ1 production and secretion to a level only observed in co-cultures of MSC-CAF, TAM and NB cells led to inhibition of NK cell-mediated NB cytotoxicity. Increased TGFβ1, NFκB and IL6 activities were also detected in NB tumors implanted in immunodeficient mice in the presence of human MSC-CAF and monocytes and in primary human NB tumors where we observed a direct correlation between pSMAD2, pSTAT3, and the presence of α-SMA positive MSC-CAF. Our results emphasize the importance of the synergistic role of TAM and CAF in promoting immune escape and resistance to immunotherapies, and the need to define the TME of tumors in patients with NB in order to improve immunotherapies. Citation Format: Kevin Louault, Tania Porras, Meng-Hua Lee, Sakunthala Muthugounder, Rebekah Kennedy, Laurence Sarte, Gerardo E. Fernandez, Bruce Pawel, Hiroyuki Shimada, Shahab Asgharzadeh, Yves A. Declerck. Cancer-associated fibroblasts and tumor-associated macrophages cooperate to promote TGF-β1-dependent NFkB activation and IL6 production and immune escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3123.

Neuroblastoma (NBL) accounts for a disproportionate number of deaths among childhood malignancies despite intensive multimodal therapy that includes antibody targeting disialoganglioside GD2, a NBL antigen. Unfortunately, resistance to anti-GD2 immunotherapy is frequent and we aimed to investigate mechanisms of resistance in NBL. GD2 expression was quantified by flow cytometry and anti-GD2 antibody internalization was measured using real-time microscopy in 20 human NBL cell lines. Neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were performed on a subset of the cell lines (n = 12), and results were correlated with GD2 expression and antibody internalization. GD2 was expressed on 19 of 20 NBL cell lines at variable levels, and neutrophil-mediated ADCC was observed only in GD2-expressing cell lines. We found no correlation between level of GD2 expression and sensitivity to neutrophil-mediated ADCC, suggesting that GD2 expression of many cell lines was above a threshold required for maximal ADCC, such that expression level could not be used to predict subsequent cytotoxicity. Instead, anti-GD2 antibody internalization, a process that occurred universally but differentially across GD2-expressing NBL cell lines, was inversely correlated with ADCC. Treatment with endocytosis inhibitors EIPA, chlorpromazine, MBCD, and cytochalasin-D showed potential to inhibit antibody internalization; however, only MBCD resulted in significantly increased sensitivity to neutrophil-mediated ADCC in 4 of 4 cell lines in vitro. Our data suggest that antibody internalization may represent a novel mechanism of immunotherapy escape by NBL and provide proof-of-principle that targeting pathways involved in antibody internalization may improve the efficacy of anti-GD2 immunotherapies.