Neuroblastoma (NB) is a heterogeneous extracranial tumor occurring in childhood. A distinctive feature of NB tumors is their neuroendocrine ability to secrete catecholamines, which in turn, via β-adrenergic receptors ligation, may affect different signaling pathways in tumor microenvironment (TME). It was previously demonstrated that specific antagonism of β3-adrenergic receptor (β3-AR) on NB tumor cells affected tumor growth and progression. Here, in a murine syngeneic model of NB, we aimed to investigate whether the β3-AR modulation influenced the host immune system response against tumor. Results demonstrated that β3-AR antagonism lead to an immune response reactivation, partially dependent on the PD-1/PD-L1 signaling axis involvement. Indeed, β3-AR blockade on tumor-infiltrating lymphocytes (TILs) dampened their ability to secrete IFN-γ, which in turn reduced the PD-L1 expression, caused by TILs infiltration, on NB tumor cells. Further investigations, through a genomic analysis on NB patients, showed that high ADRB3 gene expression correlates with worse clinical outcome compared to the low expression group, and that ADRB3 gene expression affects different immune-related pathways. Overall, results indicate that β3-AR in NB TME is able to modulate the interaction between tumor and host immune system, and that its antagonism hits multiple pro-tumoral signaling pathways.

In this study we aimed to investigate the role of the β3-adrenergic receptor (β3-AR) in regulating the immune system response against neuroblastoma (NB) tumor. NB is a very heterogeneous extracranial tumor occurring in childhood. A distinctive feature of NB tumors is their neuroendocrine ability to secrete catecholamines, which in turn, via β-adrenergic receptors ligation, may affect different signaling pathways in the tumor microenvironment (TME). We previously demonstrated that the specific antagonism of the β3-AR on NB tumor cells affected tumor growth and progression. Here, in a murine syngeneic model of NB (A/J mice inoculated with the murine neuroblastoma cell line Neuro-2a), the β3-AR blockade ability to influence the number of immunoreactive and/or immunosuppressive cells in TME was investigated through flow cytometry analysis. Moreover, the involvement of the immune-checkpoint signaling axis PD-1/PD-L1 in mediating the anti-tumoral effects brought by a β3-AR antagonist administration was analyzed. Results demonstrated that β3-AR antagonism leads to an immune response reactivation, partially dependent on the PD-1/PD-L1 signaling axis involvement. Indeed, β3-AR antagonism was able to increase the number of immune reactive CD8+, natural killer (NK) and dendritic cells (DCs), and to decrease the number of immune suppressive regulatory T cells (Treg) and Myeloid-derived suppressor cells (MDSC) in tumor mass. Moreover, β3-AR blockade on tumor infiltrating lymphocytes (TILs) dampened their ability to secrete IFN-γ, which in turn reduced the PD-L1 expression on NB tumor cells. Further investigations, through a genomic analysis on NB patients, showed that high ADRB3 gene expression correlates negatively with the patient’s clinical outcome compared to the low expression group, and that ADRB3 gene expression is also related to the expression of different immune-checkpoints. Overall, results indicate that β3-AR in NB TME is able to modulate the interaction between tumor and host immune system, and that its antagonism hits multiple pro-tumoral signaling pathways. Citation Format: Gennaro Bruno, Nicoletta Nastasi, Angela Subbiani, Alessia Boaretto, Annalisa Tondo, Claudio Favre, Maura Calvani. β3-adrenergic receptor sustains IFN-γ-dependent PD-L1 expression and impairs anti-tumor immunity in neuroblastoma [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 3887.

Osteosarcoma is a primary malignant bone tumor highly aggressive and metastasizing, composed of mesenchymal cells producing osteoid and immature matrix. Osteosarcoma is the most frequent bone tumor and it mainly affects children and adolescents. The main treatment option is a combination of surgery and chemotherapy. Research into new therapeutic strategies to improve the quality of life of patients is currently underway. The development of the anti-tumor immunostimulant drug Mifamurtide is part of this research. There are very conflicting opinions on the therapeutic validity of this drug. Therefore, we tested experimentally the effectiveness of Mifamurtide in three cell lines of osteosarcoma (MG-63, HOS, 143B) with different grades of malignancy, grown in co-culture with macrophage to allow the correct internalization of the drug. Through in vitro experiments, we analyzed the effects of Mifamurtide on tumor cell viability, on cellular signaling pathway and on macrophage polarization. Results show that Mifamurtide loses its effectiveness as the tumor malignity increases. The most aggressive tumor cells are not affected by the therapy, probably because they implement methods of resistance to apoptosis. Furthermore, they would be able to affect the tumor microenvironment to their own advantage. To better understand Mifamurtide impact on tumor microenvironment, we also performed a cytokine assay on the culture medium in which the cells grew. The most significant results were identified in the interleukine-10 (IL-10): IL-10 is expressed in higher quantities in the highest-grade osteosarcoma cells. This anti-inflammatory cytokine can inhibit the synthesis of pro-inflammatory cytokine and suppresses the antigen presentation capacity of APC cells. This allows cancer cells to evade immune surveillance mechanisms. In vivo and in vitro results show that the synergic use of an anti-IL-10 antibody in combination with Mifamurtide can improve its efficacy. Here, we provide experimental evidence that the addition of an anti-IL-10 antibody to Mifamurtide causes a significant death rate in more aggressive osteosarcoma cells and lower metastasis, compared to Mifamurtide only. In conclusion, considering our data, it could be proposed a new treatment protocol for metastatic osteosarcoma that includes the use of an anti-IL10 antibody integrated with the administration of Mifamurtide to increase its effectiveness in this clinical context. Citation Format: Nicoletta Nastasi, Angela Subbiani, Maria Letizia Taddei, Lucia Scala, Angela Tamburini, Claudio Favre, Maura Calvani. Use of an anti-IL-10 antibody to improve Mifamurtide efficacy on aggressive osteosarcoma cells [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 2007.

Neuroblastoma (NB) is a heterogeneous, often aggressive, extracranial tumor and the most common malignancy to be diagnosed in the first year of life. It has been already demonstrated that the specific antagonism of the β3-adrenergic receptor (β3-AR) on NB tumor cells affects tumor growth. To confirm the results obtained with the pharmacological approach on the crucial role played by the β3-AR receptor in influencing different pro-tumoral signaling pathways, here we investigated the effect of CRISPR/Cas9-mediated knock-out of β3-AR gene (ADRB3) on human NB cell line BE(2)-C in vitro and in vivo. Inoculation of BE(2)-C β3-AR−/- cells in athymic nude mice (NU(N-Cr)-Foxn1nu) showed a strong reduction of NB tumor growth compared to wild type cells, and in some cases β3-AR knock-out tumor cells failed in producing a tumor mass. RNA Sequencing on these tumor masses showed alterations of signaling pathways related to various tumor mechanisms, such as proliferation, cell motility and cell differentiation. Furthermore, we evaluated by flow cytometry the expression of β3-AR in circulating tumor cells (CTCs) from NB patients with different degrees of disease. Following depletion with anti-CD45 magnetic beads, tumor cells were identified through the expression of specific tumor markers, CD56 and GD2. Results showed that a high expression of β3-AR correlates with poor prognosis, characterized by metastatic or recurrent tumors, compared to patients with a favorable clinical outcome that instead show a low expression of β3-AR. Overall, our results demonstrate the β3-AR involvement in supporting NB tumor growth and suggest a possible role in regulating the metastatic potential of this tumor. Citation Format: Alessia Boaretto, Gennaro Bruno, Nicoletta Nastasi, Claudio Favre, Maura Calvani. β3-adrenergic receptor correlates with tumor growth and progression 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 6721.